Methods for identifying risk of breast cancer and treatments thereof

ABSTRACT

Provided herein are methods for identifying risk of breast cancer in a subject and/or a subject at risk of breast cancer, reagents and kits for carrying out the methods, methods for identifying candidate therapeutics for treating breast cancer, and therapeutic methods for treating breast cancer in a subject. These embodiments are based upon an analysis of polymorphic variations in nucleotide sequences within the human genome.

RELATED PATENT APPLICATIONS

This patent application claims the benefit of provisional patent application no. 60/429,136 filed Nov. 25, 2002 and provisional patent application no. 60/490,234 filed Jul. 24, 2003, having attorney docket number 524593004100 and 524593004101, respectively. Each of these provisional patent applications names Richard B. Roth et al. as inventors and is hereby incorporated herein by reference in its entirety, including all drawings and cited publications and documents.

FIELD OF THE INVENTION

The invention relates to genetic methods for identifying risk of breast cancer and treatments that specifically target the disease.

BACKGROUND

Breast cancer is the third most common cancer, and the most common cancer in women, as well as a cause of disability, psychological trauma, and economic loss. Breast cancer is the second most common cause of cancer death in women in the United States, in particular for women between the ages of 15 and 54, and the leading cause of cancer-related death (Forbes, Seminars in Oncology, vol. 24(1), Suppl 1, 1997: pp. S1-20-S1-35). Indirect effects of the disease also contribute to the mortality from breast cancer including consequences of advanced disease, such as metastases to the bone or brain. Complications arising from bone marrow suppression, radiation fibrosis and neutropenic sepsis, collateral effects from therapeutic interventions, such as surgery, radiation, chemotherapy, or bone marrow transplantation-also contribute to the morbidity and mortality from this disease.

While the pathogenesis of breast cancer is unclear, transformation of normal breast epithelium to a malignant phenotype may be the result of genetic factors, especially in women under thirty (Miki, et al., Science, 266: 66-71 (1994)). However, it is likely that other, non-genetic factors also have a significant effect on the etiology of the disease. Regardless of its origin, breast cancer morbidity increases significantly if it is not detected early in its progression. Thus, considerable efforts have focused on the elucidation of early cellular events surrounding transformation in breast tissue. Such efforts have led to the identification of several potential breast cancer markers. For example, alleles of the BRCA1 and BRCA2 genes have been linked to hereditary and early-onset breast cancer (Wooster, et al., Science, 265: 2088-2090 (1994)). However, BRCA1 is limited as a cancer marker because BRCA1 mutations fail to account for the majority of breast cancers (Ford, et al., British J. Cancer, 72: 805-812 (1995)). Similarly, the BRCA2 gene, which has been linked to forms of hereditary breast cancer, accounts for only a small portion of total breast cancer cases.

SUMMARY

It has been discovered that certain polymorphic variations in human genomic DNA are associated with the occurrence of breast cancer. In particular, polymorphic variants in loci containing GP6, LAMA4, CHGB/C20orf154 (hereafter referred to as “CHGB”), LOC338749 and IN/LOC351327 (hereafter referred to as “TTN”) regions in human genomic DNA have been associated with risk of breast cancer.

Thus, featured herein are methods for identifying a subject at risk of breast cancer and/or a risk of breast cancer in a subject, which comprises detecting the presence or absence of one or more polymorphic variations associated with breast cancer in genomic regions described herein in a human nucleic acid sample. In an embodiment, two or more polymorphic variations are detected in two or more regions selected from the group consisting of GP6, LAMA4, CHGB, LOC338749 and TTN. In certain embodiments, 3 or fewer, or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or fewer polymorphic variants are detected.

Also featured are nucleic acids that include one or more polymorphic variations associated with the occurrence of breast cancer, as well as polypeptides encoded by these nucleic acids. Further, provided is a method for identifying a subject at risk of breast cancer and then prescribing to the subject a breast cancer detection procedure, prevention procedure and/or a treatment procedure. In addition, provided are methods for identifying candidate therapeutic molecules for treating breast cancer and related disorders, as well as methods for treating breast cancer in a subject by diagnosing breast cancer in the subject and treating the subject with a suitable treatment, such as administering a therapeutic molecule.

Also provided are compositions comprising a breast cancer cell and/or GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid with a RNAi, siRNA, antisense DNA or RNA, or ribozyme nucleic acid designed from a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence. In an embodiment, the nucleic acid is designed from a GP6, LAMA4, CHGB, LOC338749 or TIN nucleotide sequence that includes one or more breast cancer associated polymorphic variations, and in some instances, specifically interacts with such a nucleotide sequence. Further, provided are arrays of nucleic acids bound to a solid surface, in which one or more nucleic acid molecules of the array have a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence, or a fragment or substantially identical nucleic acid thereof, or a complementary nucleic acid of the foregoing. Featured also are compositions comprising a breast cancer cell and/or a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide, with an antibody that specifically binds to the polypeptide. In an embodiment, the antibody specifically binds to an epitope in the polypeptide that includes a non-synonymous amino acid modification associated with breast cancer (e.g., results in an amino acid substitution in the encoded polypeptide associated with breast cancer). In certain embodiments, the antibody specifically binds to an epitope that comprises a a lysine at amino acid position 237 of SEQ ID NO: 12, a proline at amino acid position 413 of SEQ ID NO: 16 or a glutamine at amino acid position 63 of SEQ ID NO: 16.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1Y show a genomic nucleotide sequence for an GP6 region. The genomic nucleotide sequence is set forth in SEQ ID NO: 1. The following nucleotide representations are used throughout: “A” or “a” is adenosine, adenine, or adenylic acid; “C” or “c” is cytidine, cytosine, or cytidylic acid; “G” or “g” is guanosine, guanine, or guanylic acid; “T” or “t” is thymidine, thymine, or thymidylic acid; and “I” or “i” is inosine, hypoxanthine, or inosinic acid. Exons are indicated in italicized lower case type, introns are depicted in normal text lower case type, and polymorphic sites are depicted in bold upper case type. SNPs are designated by the following convention: “R” represents A or G, “M” represents A or C; “W” represents A or T; “Y” represents C or T; “S” represents C or G; “K” represents G or T; “V” represents A, C or G; “H” represents A, C, or T; “D” represents A, G, or T; “B” represents C, G, or T; and “N” represents A, G, C, or T.

FIGS. 2A-2Y show a genomic nucleotide sequence of a LAMA4 region. The genomic nucleotide sequence is set forth in SEQ ID NO: 2.

FIGS. 3A-3X show a genomic nucleotide sequence of a CHGB region. The genomic nucleotide sequence is set forth in SEQ ID NO: 3.

FIGS. 4A-4Y show a genomic nucleotide sequence of a LOC338749 region. The genomic nucleotide sequence is set forth in SEQ ID NO: 4.

FIGS. 5A-5Z show a genomic nucleotide sequence of a TTN region. The genomic nucleotide sequence is set forth in SEQ ID NO: 5.

FIGS. 6A-6B show three coding nucleotide sequences (cDNA) for GP6. The nucleotide sequence are set forth as SEQ ID NO: 6-8.

FIGS. 7A-7B show a coding nucleotide sequence (cDNA) for LAMA4. The nucleotide sequence is set forth in SEQ ID NO: 9.

FIG. 8 shows a coding nucleotide sequence (cDNA) for CHGB. The nucleotide sequence is set forth in SEQ ID NO: 10.

FIG. 9 shows a coding nucleotide sequence (cDNA) for TTN. The nucleotide sequence is set forth in SEQ ID NO: 11.

FIGS. 10A-10C show three GP6 polypeptide amino acid sequences, which are set forth as SEQ ID NO: 12-14.

FIGS. 11A-11B show an amino acid sequence for a LAMA4 polypeptide, which is set forth in SEQ ID NO: 15.

FIG. 12 shows an amino acid sequence for a CHGB polypeptide, which is set forth in SEQ ID NO: 16.

FIG. 13 shows an amino acid sequence for a TTN polypeptide, which is set forth in SEQ ID NO: 17.

FIGS. 14-18 show proximal SNPs in GP6, LAMA4, CHGB, LOC338749 and TTN loci in genomic DNA. The position of each SNP on the chromosome is shown on the x-axis and the y-axis provides the negative logarithm of the p-value comparing the estimated allele to that of the control group. Also shown in the figure are exons and introns of the genes in the approximate chromosomal positions. The figure indicates that polymorphic variants associated with breast cancer are in linkage disequilibrium in the following regions: the region spanning positions 185-8377 in SEQ ID NO: 1; the region spanning positions 506-95220 in SEQ ID NO: 2; the region spanning positions 5621-82574 in SEQ ID NO: 3; the region spanning positions 16120-55750 in SEQ ID NO: 4 or the region spanning positions 12473-96589 in SEQ ID NO: 5.

DETAILED DESCRIPTION

It has been discovered that polymorphic variations in the GP6, LAMA4, CHGB, LOC338749 and TTN regions described herein are associated with an increased risk of breast cancer.

The gene GP6 (glycoprotein VI (platelet)) is also known as GPIV and GPVI. GP6 has been mapped to chromosomal position 19q13.4. Glycoprotein VI (GP6) is a 58-kD platelet membrane glycoprotein that plays a crucial role in the collagen-induced activation and aggregation of platelets. Upon injury to the vessel wall and subsequent damage to the endothelial lining, exposure of the subendothelial matrix to blood flow results in deposition of platelets. Collagen fibers are the most thrombogenic macromolecular components of the extracellular matrix, with collagen types I, III, and VI being the major forms found in blood vessels. Platelet interaction with collagen occurs as a 2-step procedure: (1) the initial adhesion to collagen is followed by (2) an activation step leading to platelet secretion, recruitment of additional platelets, and aggregation. In physiologic conditions, the resulting platelet plug is the initial hemostatic event limiting blood loss. However, exposure of collagen after rupture of atherosclerotic plaques is a major stimulus of thrombus formation associated with myocardial infarction or stroke. Based on the fact that GP VI is coupled to the Fc receptor-gamma chain (FCER1G; 147139) and thus should share homology with the FcR chains, they have been identified as human and mouse GP VI genes. They belong to the immunoglobulin superfamily and share 64% amino acid sequence homology. Functional evidence demonstrating the identity of the recombinant protein with GP VI was provided by binding to its natural ligand collagen; binding to convulxin (a GP VI-specific ligand from snake venom); binding of anti-GP VI IgG isolated from a patient; and association to the FcR-gamma chain.

The gene LAMA4 (laminin, alpha 4) is also known as laminin, alpha 4 precursor and LAMA3. LAMA4 has been mapped to chromosomal position 6q21. Laminins, a family of extracellular matrix glycoproteins, are the major noncollagenous constituent of basement membranes. They have been implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis. Laminins are composed of 3 non identical chains: laminin alpha, beta and gamma (formerly A, B1, and B2, respectively) and they form a cruciform structure consisting of 3 short arms, each formed by a different chain, and a long arm composed of all 3 chains. Each laminin chain is a multidomain protein encoded by a distinct gene. Several isoforms of each chain have been described. Different alpha, beta and gamma chain isomers combine to give rise to different heterotrimeric laminin isoforms which are designated by Arabic numerals in the order of their discovery, i.e. alpha1beta1gamma1 heterotrimer is laminin 1. The biological functions of the different chains and trimer molecules are largely unknown, but some of the chains have been shown to differ with respect to their tissue distribution, presumably reflecting diverse functions in vivo. This gene encodes the alpha chain isoform laminin, alpha 4. The domain structure of alpha 4 is similar to that of alpha 3, both of which resemble truncated versions of alpha 1 and alpha 2, in that approximately 1,200 residues at the N-terminus (domains IV, V and VI) have been lost. Laminin, alpha 4 contains the C-terminal G domain which distinguishes all alpha chains from the beta and gamma chains. The RNA analysis from adult and fetal tissues revealed developmental regulation of expression, however, the exact function of laminin, alpha 4 is not known. Tissue-specific utilization of alternative polyA-signal has been described in literature. Also, alternative splicing involving the first intron in the 5′ UTR, and laminin alpha 4 like isoforms have been noted, however, the full-length nature of these products is not known.

The gene (chromogranin B (secretogranin 1) is also known as SCG1. CHGB has been mapped to chromosomal position 20pter-p12. Chromogranin B is a tyrosine-sulfated secretory protein found in a wide variety of peptidergic endocrine cells. Chromogranin functions as a neuroendocrine secretory granule protein which likely is the precursor for other biologically active peptides.

The gene V20orf124 is also known as MCM8 minichromosome maintenance deficient 8 (S. cerevisiae), MGC4816, MGC12866, dJ967N21.5 and DNA replication licensing factor MCM8. C20Orf124 has been mapped to chromosomal position 20p12.3. The protein encoded by this gene is one of the highly conserved mini-chromosome maintenance proteins (MCM) that are essential for the initiation of eukaryotic genome replication. The hexameric protein complex formed by the MCM proteins is a key component of the pre-replication complex (pre RC) and may be involved in the formation of replication forks and in the recruitment of other DNA replication related proteins. This protein contains the central domain that is conserved among the MCM proteins. This protein has been shown to co-immunoprecipitate with MCM4, 6 and 7, which suggests that it may interact with other MCM proteins and play a role in DNA replication. Alternatively spliced transcript variants encoding distinct isoforms have been described.

The gene LOC338749 has been mapped to chromosomal position 11p15.3.

The gene TTN(titin) is also known as TMD, CMD1G, CMPD4, FLJ32040, connectin, CMH9, included cardiomyopathy, dilated 1G (autosomal dominant). TTN has been mapped to chromosomal position 2q31. This gene encodes a large abundant protein of striated muscle. The product of this gene is divided into two regions, a N-terminal I-band and a C-terminal A-band. The I-band, which is the elastic part of the molecule, contains two regions of tandem immunoglobulin domains on either side of a PEVK region that is rich in proline, glutamate, valine and lysine. The A-band, which is thought to act as a protein-ruler, contains a mixture of immunoglobulin and fibronectin repeats, and possesses kinase activity. A N-terminal Z-disc region and a C-terminal M-line region bind to the Z-line and M-line of the sarcomere respectively so that a single titin molecule spans half the length of a sarcomere. Titin also contains binding sites for muscle associated proteins so it serves as an adhesion template for the assembly of contractile machinery in muscle cells. It has also been identified as a structural protein for chromosomes. Considerable variability exists in the I-band, the M-line and the Z-disc regions of titin. Variability in the I-band region contributes to the differences in elasticity of different titin isoforms and, therefore, to the differences in elasticity of different muscle types. Of the many titin variants identified, complete transcript information is available for five. Mutations in this gene are associated with familial hypertrophic cardiomyopathy 9 and autoantibodies to titin are produced in patients with the autoimmune disease scleroderma.

Breast Cancer and Sample Selection

Breast cancer is typically described as the uncontrolled growth of malignant breast tissue. Breast cancers arise most commonly in the lining of the milk ducts of the breast (ductal carcinoma), or in the lobules where breast milk is produced (lobular carcinoma). Other forms of breast cancer include Inflammatory Breast Cancer and Recurrent Breast Cancer. Inflammatory breast cancer is a rare, but very serious, aggressive type of breast cancer. The breast may look red and feel warm with ridges, welts, or hives on the breast; or the skin may look wrinkled. It is sometimes misdiagnosed as a simple infection. Recurrent disease means that the cancer has come back after it has been treated. It may come back in the breast, in the soft tissues of the chest (the chest wall), or in another part of the body.

As used herein, the term “breast cancer” refers to a condition characterized by anomalous rapid proliferation of abnormal cells in one or both breasts of a subject. The abnormal cells often are referred to as 37 neoplastic cells,” which are transformed cells that can form a solid tumor. The term “tumor” refers to an abnormal mass or population of cells (i.e. two or more cells) that result from excessive or abnormal cell division, whether malignant or benign, and pre-cancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cellular proliferation, they can invade surrounding tissues and can metastasize. In breast cancer, neoplastic cells may be identified in one or both breasts only and not in another tissue or organ, in one or both breasts and one or more adjacent tissues or organs (e.g. lymph node), or in a breast and one or more non-adjacent tissues or organs to which the breast cancer cells have metastasized.

The term “invasion” as used herein refers to the spread of cancerous cells to adjacent surrounding tissues. The term “invasion” often is used synonymously with the term “metastasis,” which as used herein refers to a process in which cancer cells travel from one organ or tissue to another non-adjacent organ or tissue. Cancer cells in the breast(s) can spread to tissues and organs of a subject, and conversely, cancer cells from other organs or tissue can invade or metastasize to a breast. Cancerous cells from the breast(s) may invade or metastasize to any other organ or tissue of the body. Breast cancer cells often invade lymph node cells and/or metastasize to the liver, brain and/or bone and spread cancer in these tissues and organs. Breast cancers can spread to other organs and tissues and cause lung cancer, prostate cancer, colon cancer, ovarian cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, bladder cancer, hepatoma, colorectal cancer, uterine cervical cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, skin cancer, melanoma, ovarian cancer, neuroblastoma, myeloma, various types of head and neck cancer, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma and peripheral neuroepithelioma, and other carcinomas, lymphomas, blastomas, sarcomas, and leukemias.

Breast cancers arise most commonly in the lining of the milk ducts of the breast (ductal carcinoma), or in the lobules where breast milk is produced (lobular carcinoma). Other forms of breast cancer include Inflammatory Breast Cancer and Recurrent Breast Cancer. Inflammatory Breast Cancer is a rare, but very serious, aggressive type of breast cancer. The breast may look red and feel warm with ridges, welts, or hives on the breast; or the skin may look wrinkled. It is sometimes misdiagnosed as a simple infection. Recurrent disease means that the cancer has come back after it has been treated. It may come back in the breast, in the soft tissues of the chest (the chest wall), or in another part of the body. As used herein, the term “breast cancer” may include both Inflammatory Breast Cancer and Recurrent Breast Cancer.

In an effort to detect breast cancer as early as possible, regular physical exams and screening mammograms often are prescribed and conducted. A diagnostic mammogram often is performed to evaluate a breast complaint or abnormality detected by physical exam or routine screening mammography. If an abnormality seen with diagnostic mammography is suspicious, additional breast imaging (with exams such as ultrasound) or a biopsy may be ordered. A biopsy followed by pathological (microscopic) analysis is a definitive way to determine whether a subject has breast cancer. Excised breast cancer samples often are subjected to the following analyses: diagnosis of the breast tumor and confirmation of its malignancy; maximum tumor thickness; assessment of completeness of excision of invasive and in situ components and microscopic measurements of the shortest extent of clearance; level of invasion; presence and extent of regression; presence and extent of ulceration; histological type and special variants; pre-existing lesion; mitotic rate; vascular invasion; neurotropism; cell type; tumor lymphocyte infiltration; and growth phase.

The stage of a breast cancer can be classified as a range of stages from Stage 0 to Stage IV based on its size and the extent to which it has spread. The following table summarizes the stages: TABLE A Lymph Stage Tumor Size Node Involvement Metastasis (Spread) I Less than 2 cm No No II Between 2-5 cm No or in same side of No breast III More than 5 cm Yes, on same side of No breast IV Not applicable Not applicable Yes

Stage 0 cancer is a contained cancer that has not spread beyond the breast ductal system. Fifteen to twenty percent of breast cancers detected by clinical examinations or testing are in Stage 0 (the earliest form of breast cancer). Two types of Stage 0 cancer are lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS). LCIS indicates high risk for breast cancer. Many physicians do not classify LCIS as a malignancy and often encounter LCIS by chance on breast biopsy while investigating another area of concern. While the microscopic features of LCIS are abnormal and are similar to malignancy, LCIS does not behave as a cancer (and therefore is not treated as a cancer). LCIS is merely a marker for a significantly increased risk of cancer anywhere in the breast. However, bilateral simple mastectomy may be occasionally performed if LCIS patients have a strong family history of breast cancer. In DCIS the cancer cells are confined to milk ducts in the breast and have not spread into the fatty breast tissue or to any other part of the body (such as the lymph nodes). DCIS may be detected on mammogram as tiny specks of calcium (known as microcalcifications) 80% of the time. Less commonly DCIS can present itself as a mass with calcifications (15% of the time); and even less likely as a mass without calcifications (<5% of the time). A breast biopsy is used to confirm DCIS. A standard DCIS treatment is breast-conserving therapy (BCT), which is lumpectomy followed by radiation treatment or mastectomy. To date, DCIS patients have chosen equally among lumpectomy and mastectomy as their treatment option, though specific cases may sometimes favor lumpectomy over mastectomy or vice versa.

In Stage 1, the primary (original) cancer is 2 cm or less in diameter and has not spread to the lymph nodes. In Stage IIA, the primary tumor is between 2 and 5 cm in diameter and has not spread to the lymph nodes. In Stage IIB, the primary tumor is between 2 and 5 cm in diameter and has spread to the axillary (underarm) lymph nodes; or the primary tumor is over 5 cm and has not spread to the lymph nodes. In Stage IIIA, the primary breast cancer of any kind that has spread to the axillary (underarm) lymph nodes and to axillary tissues. In Stage IIIB, the primary breast cancer is any size, has attached itself to the chest wall, and has spread to the pectoral (chest) lymph nodes. In Stage IV, the primary cancer has spread out of the breast to other parts of the body (such as bone, lung, liver, brain). The treatment of Stage IV breast cancer focuses on extending survival time and relieving symptoms.

Based in part upon selection criteria set forth above, individuals having breast cancer can be selected for genetic studies. Also, individuals having no history of cancer or breast cancer often are selected for genetic studies. Other selection criteria can include: a tissue or fluid sample is derived from an individual characterized as Caucasian; the sample was derived from an individual of German paternal and maternal descent; the database included relevant phenotype information for the individual; case samples were derived from individuals diagnosed with breast cancer; control samples were derived from individuals free of cancer and no family history of breast cancer; and sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study. Phenotype information included pre- or post-menopausal, familial predisposition, country or origin of mother and father, diagnosis with breast cancer (date of primary diagnosis, age of individual as of primary diagnosis, grade or stage of development, occurrence of metastases, e.g., lymph node metastases, organ metastases), condition of body tissue (skin tissue, breast tissue, ovary tissue, peritoneum tissue and myometrium), method of treatment (surgery, chemotherapy, hormone therapy, radiation therapy).

Provided herein is a set of blood samples and a set of corresponding nucleic acid samples isolated from the blood samples, where the blood samples are donated from individuals diagnosed with breast cancer. The sample set often includes blood samples or nucleic acid samples from 100 or more, 150 or more, or 200 or more individuals having breast cancer, and sometimes from 250 or more, 300 or more, 400 or more, or 500 or more individuals. The individuals can have parents from any place of origin, and in an embodiment, the set of samples are extracted from individuals of German paternal and German maternal ancestry. The samples in each set may be selected based upon five or more criteria and/or phenotypes set forth above.

Polymorphic Variants Associated with Breast Cancer

A genetic analysis provided herein linked breast cancer with polymorphic variants in the GP6, LAMA4, CHGB, LOC338749 and TTN regions of the human genome disclosed herein. As used herein, the term “polymorphic site” refers to a region in a nucleic acid at which two or more alternative nucleotide sequences are observed in a significant number of nucleic acid samples from a population of individuals. A polymorphic site may be a nucleotide sequence of two or more nucleotides, an inserted nucleotide or nucleotide sequence, a deleted nucleotide or nucleotide sequence, or a microsatellite, for example. A polymorphic site that is two or more nucleotides in length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, where all or some of the nucleotide sequences differ within the region. A polymorphic site is often one nucleotide in length, which is referred to herein as a “single nucleotide polymorphism” or a “SNP.”

Where there are two, three, or four alternative nucleotide sequences at a polymorphic site, each nucleotide sequence is referred to as a “polymorphic variant” or “nucleic acid variant.” Where two polymorphic variants exist, for example, the polymorphic variant represented in a minority of samples from a population is sometimes referred to as a “minor allele” and the polymorphic variant that is more prevalently represented is sometimes referred to as a “major allele.” Many organisms possess a copy of each chromosome (e.g., humans), and those individuals who possess two major alleles or two minor alleles are often referred to as being “homoygous” with respect to the polymorphism, and those individuals who possess one major allele and one minor allele are normally referred to as being “heterozygous” with respect to the polymorphism. Individuals who are homozygous with respect to one allele are sometimes predisposed to a different phenotype as compared to individuals who are heterozygous or homozygous with respect to another allele.

Furthermore, a genotype or polymorphic variant may be expressed in terms of a “haplotype,” which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population. For example, two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation. Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.

As used herein, the term “phenotype” refers to a trait which can be compared between individuals, such as presence or absence of a condition, a visually observable difference in appearance between individuals, metabolic variations, physiological variations, variations in the function of biological molecules, and the like. An example of a phenotype is occurrence of breast cancer.

Researchers sometimes report a polymorphic variant in a database without determining whether the variant is represented in a significant fraction of a population. Because a subset of these reported polymorphic variants are not represented in a statistically significant portion of the population, some of them are sequencing errors and/or not biologically relevant. Thus, it is often not known whether a reported polymorphic variant is statistically significant or biologically relevant until the presence of the variant is detected in a population of individuals and the frequency of the variant is determined. Methods for detecting a polymorphic variant in a population are described herein, specifically in Example 2. A polymorphic variant is statistically significant and often biologically relevant if it is represented in 5% or more of a population, sometimes 10% or more, 15% or more, or 20% or more of a population, and often 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more of a population.

A polymorphic variant may be detected on either or both strands of a double-stranded nucleic acid. For example, a thymine at a particular position in SEQ ID NO: 1 can be reported as an adenine from the complementary strand. Also, a polymorphic variant may be located within an intron or exon of a gene or within a portion of a regulatory region such as a promoter, a 5′ untranslated region (UTR), a 3′ UTR, and in DNA (e.g., genomic DNA (gDNA) and complementary DNA (cDNA)), RNA (e.g., mRNA, tRNA, and rRNA), or a polypeptide. Polymorphic variations may or may not result in detectable differences in gene expression, polypeptide structure, or polypeptide function.

In the genetic analysis that associated breast cancer with the polymorphic variants described hereafter, samples from individuals having breast cancer and individuals not having cancer were allelotyped and genotyped. The term “genotyped” as used herein refers to a process for determining a genotype of one or more individuals, where a “genotype” is a representation of one or more polymorphic variants in a population. Genotypes may be expressed in terms of a “haplotype,” which as used herein refers to two or more polymorphic variants occurring within genomic DNA in a group of individuals within a population. For example, two SNPs may exist within a gene where each SNP position includes a cytosine variation and an adenine variation. Certain individuals in a population may carry one allele (heterozygous) or two alleles (homozygous) having the gene with a cytosine at each SNP position. As the two cytosines corresponding to each SNP in the gene travel together on one or both alleles in these individuals, the individuals can be characterized as having a cytosine/cytosine haplotype with respect to the two SNPs in the gene.

It was determined that polymorphic variations associated with an increased risk of breast cancer existed in GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequences. Polymorphic variants in and around the GP6, LAMA4, CHGB, LOC338749 and TTN loci were tested for association with breast cancer. In the GP6 locus, these included polymorphic variants at positions in SEQ ID NO: 1 selected from the group consisting of 185, 237, 641, 719, 990, 2908, 3140, 3880, 4494, 5107, 5220, 6031, 8670, 13794, 16356, 17164, 17264, 20537, 20637, 20900, 21155, 21795, 21931, 22167, 22656, 23108, 23404, 24287, 24480, 24592, 24878, 26370, 27056, 27874, 31248, 31458, 31553, 31637, 31668, 31752, 37643, 43941, 44134, 44329, 44343, 44362, 44818, 44917, 45215, 45666, 45680, 46402, 46510, 46554, 46823, 47714, 48963, 49157, 49254, 49257, 49356, 55202, 55527, 55916, 56402, 56413, 56685, 56783, 58044, 58301, 58382, 58393, 58869, 59155, 59189, 62546, 62568, 70983, 71465, 71538, 72144, 72340, 72527, 72968, 73397, 73553, 73720, 74190, 74687, 74699, 75580, 76345, 76506, 77554, 77889, 77919, 78866, 79061, 83777, 84360, 84631, 85775, 87153, 89650, 89895, 90103, 90234, 90309, 90376, 90925, 91561, 91605, 92954 and 94228. Polymorphic variants in a region spanning positions 185-8377 in SEQ ID NO: 1 in particular were associated with an increased risk of breast cancer, including polymorphic variants at positions 185, 20537, 44329, 44362, 45666, 45680, 46510, 49254, 49356, 56402, 58301, 71465, 72527, 73553, 76345 and 83777 in SEQ ID NO: 1. At these positions in SEQ ID NO: 1, a thymine at position 185, a thymine at position 20537, a cytosine at position 44329, an adenine at position 44362, a guanine at position 45666, a thymine at position 45680, a thymine at position 46510, a guanine at position 49254, a thymine at position 49356, a guanine at position 56402, a cytosine at position 58301, an adenine at position 71465, a guanine at position 72527, a guanine at position 73553, a thymine at position 76345 and an adenine at position 83777 in particular were associated with risk of breast cancer. Also, a lysine at amino acid position 237 in SEQ ID NO: 12 was associated with an increased risk of breast cancer.

In the LAMA4 locus, these included polymorphic variants at positions in SEQ ID NO: 2 selected from the group consisting of 184, 506, 3981, 7815, 7875, 10775, 10786, 11013, 11020, 11101, 14171, 14278, 16512, 16706, 18442, 20286, 21591, 22275, 25318, 27997, 29840, 31088, 31258, 32367, 32427, 33671, 38796, 41530, 41874, 44161, 47502, 51089, 51205, 53645, 54280, 57610, 57740, 60812, 60837, 64448, 65249, 65482, 66535, 66789, 67214, 68347, 69060, 70100, 70215, 73687, 73732, 74183, 74813, 78136, 79540, 79655, 79731, 82111, 82155, 83479, 84511, 85290, 90620, 91127, 92095, 92679, 94839 and 95220. Polymorphic variants in a region spanning positions 506-95220 in SEQ ID NO: 2 in particular were associated with an increased risk of breast cancer, including polymorphic variants at positions 506, 3981, 7815, 7875, 11020, 11101, 18442, 47502, 53645, 65249, 73687, 73732, 74183, 79540, 82155, 85290, 90620, 91127, 92095, 92679, 94839 and 95220 in SEQ ID NO: 2. At these positions in SEQ ID NO: 2, a cytosine at position 506, a cytosine at position 3981, a guanine at position 7815, a guanine at position 7875, a thymine at position 11020, an adenine at position 11101, an adenine at position 18442, a cytosine at position 47502, a guanine at position 53645, a thymine at position 65249, a cytosine at position 73687, an adenine at position 73732, a thymine at position 74183, a thymine at position 79540, a thymine at position 82155, a cytosine at position 85290, a guanine at position 90620, a guanine at position 91127, an adenine at position 92095, a guanine at position 92679, a guanine at position 94839 and a cytosine at position 95220 in particular were associated with increased risk of breast cancer.

In the CHGB locus, these included polymorphic variants at positions in SEQ ID NO: 3 selected from the group consisting of 186, 1332, 1893, 2786, 2962, 3377, 5522, 5621, 5889, 7531, 8268, 8923, 8988, 9117, 9448, 9494, 9628, 9640, 11072, 11150, 11379, 11692, 12056, 12104, 14160, 14836, 14980, 15165, 15315, 15624, 15796, 15939, 16581, 17045, 18501, 21800, 21966, 22134, 22181, 23028, 23312, 23573, 23858, 23888, 23990, 24073, 25330, 26473, 27958, 28421, 28804, 29322, 30819, 31956, 32592, 32818, 32880, 33244, 33845, 34272, 34931, 36870, 37790, 38708, 39135, 39919, 40166, 40985, 41049, 41935, 42775, 43807, 44254, 44814, 45249, 47599, 47807, 48555, 49249, 49293, 57566, 63587, 64560, 65432, 66291, 71331, 73344, 74159, 74564, 78194, 79128, 79393, 81579, 82574, 85309, 87076, 87844 and 90241. Polymorphic variants in a region spanning positions 5621-82574 in SEQ ID NO: 3 in particular were associated with an increased risk of breast cancer, including polymorphic variants at positions 5621, 9628, 9640, 21800, 21966, 22134, 22181, 23028, 23573, 23888, 24073, 26473, 27958, 28421, 28804, 29322, 30819, 31956, 32592, 32818, 32880, 33244, 33845, 34931, 37790, 38708, 39135, 39919, 40166, 41049, 43807, 44254, 45249, 47807,4 8555, 49249, 49293, 57566, 63587, 64560, 65432, 66291, 71331, 73344, 74159, 78194, 79128, 81579 and 82574 in SEQ ID NO: 3. At these positions in SEQ ID NO: 3, a guanine at position 5621, a guanine at position 9628, a cytosine at position 9640, a guanine at position 21800, an adenine at position 21966, a guanine at position 22134, an adenine at position 22181, a guanine at position 23028, a thymine at position 23573, a guanine at positino 23888, an adenine at position 24073, a thymine at position 26473, a cytosine at position 27958, an adenine at position 28421, a thymine at position 28804, a cytosine at position 29322, a cytosine at position 30819, a guanine at position 31956, a guanine at position 32592, a cytosine at position 32818, a thymine at position 32880, a cytosine at position 33244, an adenine at position 33845, a thymine at position 34931, a thymine at position 37790, a guanine at position 38708, a thymine at position 39135, an adenine at position 39919, a thymine at position 40166, a guanine at position 41049, a cytosine at position 43807, a guanine at position 44254, a thymine at position 45249, a guanine at position 47807, a cytosine at position 48555, an adenine at position 49249, a cytosine at position 49293, a cytosine at position 57566, a cytosine at position 63587, a thymine at position 64560, a cytosine at position 65432, a thymine at position 66291, an adenine at position 71331, a thymine at position 73344, a thymine at position 74159, an adenine at position 78194, a cytosine at position 79128, an adenine at position 81579 and a cytosine at position 82574 in particular were associated with an increased risk of breast cancer. Also, a proline at amino acid position 413 and a glutamine at amino acid position 63 in particular associated with an increased risk of breast cancer.

In the LOC338749 locus, these included polymorphic variants at positions in SEQ ID NO: 4 selected from the group consisting of 142, 693, 731, 879, 1084, 2249, 2519, 4461, 4616, 5109, 5270, 5436, 5457, 6536, 9665, 16120, 29489, 29524, 49159, 49273, 49596, 50135, 50184, 50393, 50401, 55750, 73843, 73852, 74052, 75382, 75662, 75942, 77917, 78821, 94813 and 97149. Polymorphic variants in a region spanning positions 16120-55750 in SEQ ID NO: 4 in particular were associated with an increased risk of breast cancer, including polymorphic variants at positions 16120 and 55750 in SEQ ID NO: 4. At these positions in SEQ ID NO: 4, a thymine at position 16120 and a cytosine at position 55750 in particular were associated with an increased risk of breast cancer.

In the TTN locus, these included polymorphic variants at positions in SEQ ID NO: 5 selected from the group consisting of 200, 381, 5303, 6084, 6879, 7837, 7985, 9333, 11559, 12473, 12880, 13606, 14861, 20658, 22200, 24525, 26373, 42869, 43713, 44429, 49037, 49170, 50206, 51552, 51674, 56427, 56844, 57953, 60862, 61606, 62560, 65078, 65155, 70295, 70335, 70398, 79233, 80025, 84521, 84540, 85170, 85300, 87596, 89696, 92219 and 96589. Polymorphic variants in a region spanning positions 12473-96589 in SEQ ID NO: 5 in particular were associated with an increased risk of breast cancer, including polymorphic variants at positions 12473, 20658, 24525, 49037, 49170, 51552, 51674, 70335, 84521, 87596, 92219 and 96589 in SEQ ID NO: 5. At these positions in SEQ ID NO: 5, a cytosine at position 12473, a thymine at position 20658, a cytosine at position 24525, a guanine at position 49037, an adenine at position 49170, a thymine at position 51552, a guanine at position 51674, a thymine at position 70335, a guanine at position 84521, an adenine at position 87596, an adenine at position 92219 and a thymine at position 96589 in particular were associated with an increased risk of breast cancer.

Additional Polymorphic Variants Associated with Breast Cancer

Also provided is a method for identifying polymorphic variants proximal to an incident, founder polymorphic variant associated with breast cancer. Thus, featured herein are methods for identifying a polymorphic variation associated with breast cancer that is proximal to an incident polymorphic variation associated with breast cancer, which comprises identifying a polymorphic variant proximal to the incident polymorphic variant associated with breast cancer, where the incident polymorphic variant is in a nucleotide sequence set forth in SEQ ID NO: 1-5. The nucleotide sequence often comprises a polynucleotide sequence selected from the group consisting of (a) a nucleotide sequence set forth in SEQ ID NO: 1-5; (b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1-5; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1-5 or a nucleotide sequence about 90% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1-5; and (d) a fragment of a nucleotide sequence of (a), (b), or (c), often a fragment that includes a polymorphic site associated with breast cancer. The presence or absence of an association of the proximal polymorphic variant with breast cancer then is determined using a known association method, such as a method described in the Examples hereafter. In an embodiment, the incident polymorphic variant is described in SEQ ID NO: 1-5. In another embodiment, the proximal polymorphic variant identified sometimes is a publicly disclosed polymorphic variant, which for example, sometimes is published in a publicly available database. In other embodiments, the polymorphic variant identified is not publicly disclosed and is discovered using a known method, including, but not limited to, sequencing a region surrounding the incident polymorphic variant in a group of nucleic acid samples. Thus, multiple polymorphic variants proximal to an incident polymorphic variant are associated with breast cancer using this method.

The proximal polymorphic variant often is identified in a region surrounding the incident polymorphic variant. In certain embodiments, this surrounding region is about 50 kb flanking the first polymorphic variant (e.g. about 50 kb 5′ of the first polymorphic variant and about 50 kb 3′ of the first polymorphic variant), and the region sometimes is composed of shorter flanking sequences, such as flanking sequences of about 40 kb, about 30 kb, about 25 kb, about 20 kb, about 15 kb, about 10 kb, about 7 kb, about 5 kb, or about 2 kb 5′ and 3′ of the incident polymorphic variant. In other embodiments, the region is composed of longer flanking sequences, such as flanking sequences of about 55 kb, about 60 kb, about 65 kb, about 70 kb, about 75 kb, about 80 kb, about 85 kb, about 90 kb, about 95 kb, or about 100 kb 5′ and 3′ of the incident polymorphic variant.

In certain embodiments, polymorphic variants associated with breast cancer are identified iteratively. For example, a first proximal polymorphic variant is associated with breast cancer using the methods described above and then another polymorphic variant proximal to the first proximal polymorphic variant is identified (e.g., publicly disclosed or discovered) and the presence or absence of an association of one or more other polymorphic variants proximal to the first proximal polymorphic variant with breast cancer is determined.

The methods described herein are useful for identifying or discovering additional polymorphic variants that may be used to further characterize a gene, region or loci associated with a condition, a disease (e.g., breast cancer), or a disorder. For example, allelotyping or genotyping data from the additional polymorphic variants may be used to identify a functional mutation or a region of linkage disequilibrium.

In certain embodiments, polymorphic variants identified or discovered within a region comprising the first polymorphic variant associated with breast cancer are genotyped using the genetic methods and sample selection techniques described herein, and it can be determined whether those polymorphic variants are in linkage disequilibrium with the first polymorphic variant. The size of the region in linkage disequilibrium with the first polymorphic variant also can be assessed using these genotyping methods. Thus, provided herein are methods for determining whether a polymorphic variant is in linkage disequilibrium with a first polymorphic variant associated with breast cancer, and such information can be used in prognosis methods described herein.

Isolated GP6, LAMA4, CHGB, LOC338749 or TTN Nucleic Acids

Featured herein are isolated GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acids, which include the nucleic acid having the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, nucleic acid variants, and substantially identical nucleic acids of the foregoing. Nucleotide sequences of the GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acids sometimes are referred to herein as “GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequences.” A “GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid variant” refers to one allele that may have one or more different polymorphic variations as compared to another allele in another subject or the same subject. A polymorphic variation in the GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid variant may be represented on one or both strands in a double-stranded nucleic acid or on one chromosomal complement (heterozygous) or both chromosomal complements (homozygous).

As used herein, the term “nucleic acid” includes DNA molecules (e.g., a complementary DNA (cDNA) and genomic DNA (gDNA)) and RNA molecules (e.g., mRNA, rRNA, and tRNA) and analogs of DNA or RNA, for example, by use of nucleotide analogs. The nucleic acid molecule can be single-stranded and it is often double-stranded. The term “isolated or purified nucleic acid” refers to nucleic acids that are separated from other nucleic acids present in the natural source of the nucleic acid. For example, with regard to genomic DNA, the term “isolated” includes nucleic acids which are separated from the chromosome with which the genomic DNA is naturally associated. An “isolated” nucleic acid is often free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. As used herein, the term “GP6, LAMA4, CHGB, LOC338749 or TTN gene” refers to a nucleotide sequence that encodes a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide.

Also included herein are nucleic acid fragments. These fragments typically are a nucleotide sequence identical to a nucleotide sequence in SEQ ID NO: 1-11, a nucleotide sequence substantially identical to a nucleotide sequence in SEQ ID NO: 1-11, or a nucleotide sequence that is complementary to the foregoing. The nucleic acid fragment may be identical, substantially identical or homologous to a nucleotide sequence in an exon or an intron in SEQ ID NO: 1-5, and may encode a domain or part of a domain or motif of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide, sometimes the domains set forth in FIGS. 13-18. Sometimes, the fragment comprises the polymorphic variation described herein as being associated with breast cancer. The nucleic acid fragment sometimes is 50, 100, or 200 or fewer base pairs in length, and is sometimes about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3800, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000, 130000, 140000, 150000 or 160000 base pairs in length. A nucleic acid fragment complementary to a nucleotide sequence identical or substantially identical to the nucleotide sequence of SEQ ID NO: 1-11 and hybridizes to such a nucleotide sequence under stringent conditions often is referred to as a “probe.” Nucleic acid fragments often include one or more polymorphic sites, or sometimes have an end that is adjacent to a polymorphic site as described hereafter.

An example of a nucleic acid fragment is an oligonucleotide. As used herein, the term “oligonucleotide” refers to a nucleic acid comprising about 8 to about 50 covalently linked nucleotides, often comprising from about 8 to about 35 nucleotides, and more often from about 10 to about 25 nucleotides. The backbone and nucleotides within an oligonucleotide may be the same as those of naturally occurring nucleic acids, or analogs or derivatives of naturally occurring nucleic acids, provided that oligonucleotides having such analogs or derivatives retain the ability to hybridize specifically to a nucleic acid comprising a targeted polymorphism. Oligonucleotides described herein may be used as hybridization probes or as components of prognostic or diagnostic assays, for example, as described herein.

Oligonucleotides are typically synthesized using standard methods and equipment, such as the ABI 3900 High Throughput DNA Synthesizer and the EXPEDITE™ 8909 Nucleic Acid Synthesizer, both of which are available from Applied Biosystems (Foster City, Calif.). Analogs and derivatives are exemplified in U.S. Pat. Nos. 4,469,863; 5,536,821; 5,541,306; 5,637,683; 5,637,684; 5,700,922; 5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165; 5,929,226; 5,977,296; 6,140,482; WO 00/56746; WO 01/14398, and related publications. Methods for synthesizing oligonucleotides comprising such analogs or derivatives are disclosed, for example, in the patent publications cited above and in U.S. Pat. Nos. 5,614,622; 5,739,314; 5,955,599; 5,962,674; 6,117,992; in WO 00/75372; and in related publications.

Oligonucleotides also may be linked to a second moiety. The second moiety may be an additional nucleotide sequence such as a tail sequence (e.g., a polyadenosine tail), an adapter sequence (e.g., phage M13 universal tail sequence), and others. Alternatively, the second moiety may be a non-nucleotide moiety such as a moiety which facilitates linkage to a solid support or a label to facilitate detection of the oligonucleotide. Such labels include, without limitation, a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, and the like. The second moiety may be attached to any position of the oligonucleotide, provided the oligonucleotide can hybridize to the nucleic acid comprising the polymorphism.

Uses for Nucleic Acid Sequences

Nucleic acid coding sequences depicted in SEQ ID NO: 1-11 may be used for diagnostic purposes for detection and control of polypeptide expression. Also, included herein are oligonucleotide sequences such as antisense RNA, small-interfering RNA (siRNA) and DNA molecules and ribozymes that function to inhibit translation of a polypeptide. Antisense techniques and RNA interference techniques are known in the art and are described herein.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Ribozymes may be engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences corresponding to or complementary to the nucleotide sequences set forth in SEQ ID NO: 1-11. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between fifteen (15) and twenty (20) ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

Antisense RNA and DNA molecules, siRNA and ribozymes may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

DNA encoding a polypeptide also may have a number of uses for the diagnosis of diseases, including breast cancer, resulting from aberrant expression of a target gene described herein. For example, the nucleic acid sequence may be used in hybridization assays of biopsies or autopsies to diagnose abnormalities of expression or function (e.g., Southern or Northern blot analysis, in situ hybridization assays).

In addition, the expression of a polypeptide during embryonic development may also be determined using nucleic acid encoding the polypeptide. As addressed, infra, production of functionally impaired polypeptide can be the cause of various disease states, such as breast cancer. In situ hybridizations using polynucleotide probes may be employed to predict problems related to breast cancer. Further, as indicated, infra, administration of human active polypeptide, recombinantly produced as described herein, may be used to treat disease states related to functionally impaired polypeptide. Alternatively, gene therapy approaches may be employed to remedy deficiencies of functional polypeptide or to replace or compete with dysfunctional polypeptide.

Expression Vectors, Host Cells, and Genetically Engineered Cells

Provided herein are nucleic acid vectors, often expression vectors, which contain a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid, or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors may include replication defective retroviruses, adenoviruses and adeno-associated viruses for example.

A vector can include a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vector typically includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like. Expression vectors can be introduced into host cells to produce GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides, including fusion polypeptides, encoded by GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acids.

Recombinant expression vectors can be designed for expression of GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides in prokaryotic or eukaryotic cells. For example, GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides can be expressed in E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of polypeptides in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant polypeptide; 2) to increase the solubility of the recombinant polypeptide; and 3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson, Gene 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

Purified fusion polypeptides can be used in screening assays and to generate antibodies specific for GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides. In a therapeutic embodiment, fusion polypeptide expressed in a retroviral expression vector is used to infect bone marrow cells that are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

Expressing the polypeptide in host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide is often used to maximize recombinant polypeptide expression (Gottesman, S., Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. 185: 119-128 (1990)). Another strategy is to alter the nucleotide sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., Nucleic Acids Res. 20: 2111-2118 (1992)). Such alteration of nucleotide sequences can be carried out by standard DNA synthesis techniques.

When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Recombinant mammalian expression vectors are often capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include an albumin promoter (liver-specific; Pinkert et al., Genes Dev. 1: 268-277 (1987)), lymphoid-specific promoters (Calame & Eaton, Adv. Immunol. 43: 235-275 (1988)), promoters of T cell receptors (Winoto & Baltimore, EMBO J. 8: 729-733 (1989)) promoters of immunoglobulins (Banerji et al., Cell 33: 729-740 (1983); Queen & Baltimore, Cell 33: 741-748 (1983)), neuron-specific promoters (e.g., the neurofilament promoter; Byrne & Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477 (1989)), pancreas-specific promoters (Edlund et al., Science 230: 912-916 (1985)), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are sometimes utilized, for example, the murine hox promoters (Kessel & Gruss, Science 249: 374-379 (1990)) and the α-fetopolypeptide promoter (Campes & Tilghman, Genes Dev. 3: 537-546 (1989)).

A GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid may also be cloned into an expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid cloned in the antisense orientation can be chosen for directing constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. Antisense expression vectors can be in the form of a recombinant plasmid, phagemid or attenuated virus. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) (1986).

Also provided herein are host cells that include a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid within a recombinant expression vector or GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid sequence fragments which allow it to homologously recombine into a specific site of the host cell genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell but rather also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vectors can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, transduction/infection, DEAE-dextran-mediated transfection, lipofection, or electroporation.

A host cell provided herein can be used to produce (i.e., express) a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. Accordingly, further provided are methods for producing a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide using the host cells described herein. In one embodiment, the method includes culturing host cells into which a recombinant expression vector encoding a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide has been introduced in a suitable medium such that a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide is produced. In another embodiment, the method further includes isolating a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide from the medium or the host cell.

Also provided are cells or purified preparations of cells which include a GP6, LAMA14, CHGB, LOC338749 or TTN transgene, or which otherwise misexpress GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. Cell preparations can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In certain embodiments, the cell or cells include a GP6, LAMA4, CHGB, LOC338749 or TTN transgene (e.g., a heterologous form of a GP6, LAMA4, CHGB, LOC338749 or TTN such as a human gene expressed in non-human cells). The GP6, LAMA4, CHGB, LOC338749 or TTN transgene can be misexpressed, e.g., overexpressed or underexpressed. In other embodiments, the cell or cells include a gene which misexpress an endogenous GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide (e.g., expression of a gene is disrupted, also known as a knockout). Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed GP6, LAMA4, CHGB, LOC338749 or TTN alleles or for use in drug screening. Also provided are human cells (e.g., a hematopoietic stem cells) transformed with a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid.

Also provided are cells or a purified preparation thereof (e.g., human cells) in which an endogenous GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid is under the control of a regulatory sequence that does not normally control the expression of the endogenous GP6, LAMA4, CHGB, LOC338749 or TTN gene. The expression characteristics of an endogenous gene within a cell (e.g., a cell line or microorganism) can be modified by inserting a heterologous DNA regulatory element into the genome of the cell such that the inserted regulatory element is operably linked to the endogenous GP6, LAMA4, CHGB, LOC338749 or TTN gene. For example, an endogenous GP6, LAMA4, CHGB, LOC338749 or TTN gene (e.g., a gene which is “transcriptionally silent,” not normally expressed, or expressed only at very low levels) may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO 91/06667, published on May 16,1991.

Transgenic Animals

Non-human transgenic animals that express a heterologous GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide (e.g., expressed from a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid isolated from another organism) can be generated. Such animals are useful for studying the function and/or activity of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide and for identifying and/or evaluating modulators of GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid and GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide activity. As used herein, a “transgenic animal” is a non-human animal such as a mammal (e.g., a non-human primate such as chimpanzee, baboon, or macaque; an ungulate such as an equine, bovine, or caprine; or a rodent such as a rat, a mouse, or an Israeli sand rat), a bird (e.g., a chicken or a turkey), an amphibian (e.g., a frog, salamander, or newt), or an insect (e.g., Drosophila melanogaster), in which one or more of the cells of the animal includes a GP6, LAMA4, CHGB, LOC338749 or TTN transgene. A transgene is exogenous DNA or a rearrangement (e.g., a deletion of endogenous chromosomal DNA) that is often integrated into or occurs in the genome of cells in a transgenic animal. A transgene can direct expression of an encoded gene product in one or more cell types or tissues of the transgenic animal, and other transgenes can reduce expression (e.g., a knockout). Thus, a transgenic animal can be one in which an endogenous GP6, LAMA4, CHGB, LOC338749 or TTN gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal (e.g., an embryonic cell of the animal) prior to development of the animal.

Intronic sequences and polyadenylation signals can also be included in the transgene to increase expression efficiency of the transgene. One or more tissue-specific regulatory sequences can be operably linked to a GP6, LAMA4, CHGB, LOC338749 or TTN transgene to direct expression of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide to particular cells. A transgenic founder animal can be identified based upon the presence of a GP6, LAMA4, CHGB, LOC338749 or TTN transgene in its genome and/or expression of GP6, LAMA4, CHGB, LOC338749 or TTN mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide can further be bred to other transgenic animals carrying other transgenes.

GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides can be expressed in transgenic animals or plants by introducing, for example, a nucleic acid encoding the polypeptide into the genome of an animal. In certain embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Also included is a population of cells from a transgenic animal.

GP6. LAMA4, CHGB, LOC338749 and TTN Polypeptides

Featured herein are isolated GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides, which include polypeptides having amino acid sequences set forth in SEQ ID NO: 12-17, and substantially identical polypeptides thereof. Such polypeptides sometimes are proteins or peptides. A GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide is a polypeptide encoded by a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid, where one nucleic acid can encode one or more different polypeptides. An “isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language “substantially free” means preparation of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide variant having less than about 30%, 20%, 10% and sometimes 5% (by dry weight), of non-GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide (also referred to herein as a “contaminating protein”), or of chemical precursors or non-GP6, LAMA4, CHGB, LOC338749 or TTN chemicals. When the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or a biologically active portion thereof is recombinantly produced, it is also often substantially free of culture medium, specifically, where culture medium represents less than about 20%, sometimes less than about 10%, and often less than about 5% of the volume of the polypeptide preparation. Isolated or purified GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide preparations are sometimes 0.01 milligrams or more or 0.1 milligrams or more, and often 1.0 milligrams or more and 10 milligrams or more in dry weight. In specific embodiments, a GP6 polypeptide comprises a lysine at amino acid position 237 of SEQ ID NO: 12, and a CHGB polypeptide comprises a proline at amino acid position 413 of SEQ ID NO: 16 or a glutamine at amino acid position 63 of SEQ ID NO: 16.

In another aspect, featured herein are GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides and biologically active or antigenic fragments thereof that are useful as reagents or targets in assays applicable to prevention, treatment or diagnosis of breast cancer. In another embodiment, provided herein are GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides having a GP6, LAMA4, CHGB, LOC338749 or TTN activity or activities.

Further included herein are GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide fragments. The polypeptide fragment may be a domain or part of a domain of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. The polypeptide fragment is often 50 or fewer, 100 or fewer, or 200 or fewer amino acids in length, and is sometimes 300, 400, 500, 600, 700, or 900 or fewer amino acids in length. In certain embodiments, the polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 1211 consecutive amino acids of SEQ ID NO: 12-17, or the polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids and not more than 543 consecutive amino acids of SEQ ID NO: 12-17.

GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides described herein can be used as immunogens to produce anti-GP6, LAMA4, CHGB, LOC338749 or TTN antibodies in a subject, to purify GP6, LAMA4, CHGB, LOC338749 or TTN ligands or binding partners, and in screening assays to identify molecules which inhibit or enhance the interaction of GP6, LAMA4, CHGB, LOC338749 or TTN with a GP6, LAMA4, CHGB, LOC338749 or TTN substrate. Full-length GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides and polynucleotides encoding the same may be specifically substituted for a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide fragment or polynucleotide encoding the same in any embodiment described herein.

Substantially identical polypeptides may depart from the amino acid sequences set forth in SEQ ID NO: 12-17 in different manners. For example, conservative amino acid modifications may be introduced at one or more positions in the amino acid sequences of SEQ ID NO: 12-17. A “conservative amino acid substitution” is one in which the amino acid is replaced by another amino acid having a similar structure and/or chemical function. Families of amino acid residues having similar structures and functions are well known. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Also, essential and non-essential amino acids may be replaced. A “non-essential” amino acid is one that can be altered without abolishing or substantially altering the biological function of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide, whereas altering an “essential” amino acid abolishes or substantially alters the biological function of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. Amino acids that are conserved among GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides are typically essential amino acids.

Also, GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides and polypeptide variants may exist as chimeric or fusion polypeptides. As used herein, a GP6, LAMA4, CHGB, LOC338749 or TTN “chimeric polypeptide” or “fusion polypeptide” includes a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide linked to a non-GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. A “non-GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially identical to the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide, which includes, for example, a polypeptide that is different from the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide and derived from the same or a different organism. The GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide in the fusion polypeptide can correspond to an entire or nearly entire GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or a fragment thereof. The non-GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide can be fused to the N-terminus or C-terminus of the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide.

Fusion polypeptides can include a moiety having high affinity for a ligand. For example, the fusion polypeptide can be a GST-GP6, LAMA4, CHGB, LOC338749 or TTN fusion polypeptide in which the GP6, LAMA4, CHGB, LOC338749 or TTN sequences are fused to the C-terminus of the GST sequences, or a polyhistidine-GP6, LAMA4, CHGB, LOC338749 or TTN fusion polypeptide in which the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide is fused at the N— or C-terminus to a string of histidine residues. Such fusion polypeptides can facilitate purification of recombinant GP6, LAMA4, CHGB, LOC338749 or TTN. Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide), and a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid can be cloned into an expression vector such that the fusion moiety is linked in-frame to the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. Further, the fusion polypeptide can be a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression, secretion, cellular internalization, and cellular localization of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide can be increased through use of a heterologous signal sequence. Fusion polypeptides can also include all or a part of a serum polypeptide (e.g., an IgG constant region or human serum albumin).

GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides or fragments thereof can be incorporated into pharmaceutical compositions and administered to a subject in vivo. Administration of these GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides can be used to affect the bioavailability of a GP6, LAMA4, CHGB, LOC338749 or TTN substrate and may effectively increase or decrease GP6, LAMA4, CHGB, LOC338749 or TTN biological activity in a cell or effectively supplement dysfunctional or hyperactive GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. GP6, LAMA4, CHGB, LOC338749 or TTN fusion polypeptides may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide; (ii) mis-regulation of the GP6, LAMA4, CHGB, LOC338749 or TTN gene; and (iii) aberrant post-translational modification of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. Also, GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides can be used as immunogens to produce anti-GP6, LAMA4, CHGB, LOC338749 or TTN antibodies in a subject, to purify GP6, LAMA4, CHGB, LOC338749 or TTN ligands or binding partners, and in screening assays to identify molecules which inhibit or enhance the interaction of GP6, LAMA4, CHGB, LOC338749 or TTN with a GP6, LAMA4, CHGB, LOC338749 or TTN substrate. Preferably, said GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides are used in screening assays to identify molecules which inhibit the interaction of GP6, LAMA4, CHGB, LOC338749 or TTN.

In addition, polypeptides can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins. New York, N.Y.: W. H. Freeman and Company; and Hunkapiller et al., (1984) Nature July 12-18;310(5973):105-11). For example, a relative short polypeptide fragment can be synthesized by use of a peptide synthesizer. Furthermore, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, orleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Also included are polypeptide fragments which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and the like. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; and the like.

Additional post-translational modifications include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.

Also provided are chemically modified polypeptide derivatives that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity. See U.S. Pat. No: 4,179,337. The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al. (1992) Exp Hematol. September;20(8):1028-35, reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. A polymer sometimes is attached at an amino group, such as attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, and the like), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

Substantially Identical Nucleic Acids and Polypeptides

Nucleotide sequences and polypeptide sequences that are substantially identical to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence and the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide sequences encoded by those nucleotide sequences are included herein. The term “substantially identical” as used herein refers to two or more nucleic acids or polypeptides sharing one or more identical nucleotide sequences or polypeptide sequences, respectively. Included are nucleotide sequences or polypeptide sequences that are 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (each often within a 1%, 2%, 3% or 4% variability) or more identical to the nucleotide sequences in SEQ ID NO: 1-11 or the encoded GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide amino acid sequences. One test for determining whether two nucleic acids are substantially identical is to determine the percent of identical nucleotide sequences or polypeptide sequences shared between the nucleic acids or polypeptides.

Calculations of sequence identity are often performed as follows. Sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is sometimes 30% or more, 40% or more, 50% or more, often 60% or more, and more often 70% or more, 80% or more, 90% or more, 90% or more, or 100% of the length of the reference sequence. The nucleotides or amino acids at corresponding nucleotide or polypeptide positions, respectively, are then compared among the two sequences. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the nucleotides or amino acids are deemed to be identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, introduced for optimal alignment of the two sequences.

Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers & Miller, CABIOS 4: 11-17 (1989), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Also, percent identity between two amino acid sequences can be determined using the Needleman & Wunsch, J. Mol. Biol. 48: 444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at the http address www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http address www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters often used is a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Another manner for determining if two nucleic acids are substantially identical is to assess whether a polynucleotide homologous to one nucleic acid will hybridize to the other nucleic acid under stringent conditions. As use herein, the term “stringent conditions” refers to conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are described in that reference and either can be used. An example of stringent hybridization conditions is hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 55° C. A further example of stringent hybridization conditions is hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C. Often, stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. More often, stringency conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C.

An example of a substantially identical nucleotide sequence to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence is one that has a different nucleotide sequence but still encodes the same polypeptide sequence encoded by the GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence. Another example is a nucleotide sequence that encodes a polypeptide having a polypeptide sequence that is more than 70% or more identical to, sometimes 75% or more, 80% or more, or 85% or more identical to, and often 90% or more and 95% or more identical to a polypeptide sequence encoded by a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence.

GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequences and GP6, LAMA4, CHGB, LOC338749 or TTN amino acid sequences can be used as “query sequences” to perform a search against public databases to identify other family members or related sequences, for example. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215: 403-10 (1990). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleotide sequences from SEQ ID NO: 1-11. BLAST polypeptide searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to polypeptides encoded by a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17): 3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see the http address www.ncbi.nlm.nih.gov).

A nucleic acid that is substantially identical to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence may include polymorphic sites at positions equivalent to those described herein when the sequences are aligned. For example, using the alignment procedures described herein, SNPs in a sequence substantially identical to a sequence in SEQ ID NO: 1-11 can be identified at nucleotide positions that match (i.e., align) with nucleotides at SNP positions in the nucleotide sequence of SEQ ID NO: 1-11. Also, where a polymorphic variation results in an insertion or deletion, insertion or deletion of a nucleotide sequence from a reference sequence can change the relative positions of other polymorphic sites in the nucleotide sequence.

Substantially identical nucleotide and polypeptide sequences include those that are naturally occurring, such as allelic variants (same locus), splice variants, homologs (different locus), and orthologs (different organism) or can be non-naturally occurring. Non-naturally occurring variants can be generated by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product). Orthologs, homologs, allelic variants, and splice variants can be identified using methods known in the art. These variants normally comprise a nucleotide sequence encoding a polypeptide that is 50% or more, about 55% or more, often about 70-75% or more, more often about 80-85% or more, and typically about 90-95% or more identical to the amino acid sequences of target polypeptides or a fragment thereof. Such nucleic acid molecules readily can be identified as being able to hybridize under stringent conditions to a nucleotide sequence in SEQ ID NO: 1-11 or a fragment thereof. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants of a nucleotide sequence in SEQ ID NO: 1-11 can be identified by mapping the sequence to the same chromosome or locus as the nucleotide sequence in SEQ ID NO: 1-11.

Also, substantially identical nucleotide sequences may include codons that are altered with respect to the naturally occurring sequence for enhancing expression of a target polypeptide in a particular expression system. For example, the nucleic acid can be one in which one or more codons are altered, and often 10% or more or 20% or more of the codons are altered for optimized expression in bacteria (e.g., E. coli.), yeast (e.g., S. cervesiae), human (e.g., 293 cells), insect, or rodent (e.g., hamster) cells.

Methods for Identifying Subjects at Risk of Breast Cancer and Breast Cancer Risk in a Subject

Methods for prognosing and diagnosing breast cancer in subjects are provided herein. These methods include detecting the presence or absence of one or more polymorphic variations associated with breast cancer in a nucleotide sequence set forth in SEQ ID NO: 1-5, or substantially identical sequence thereof, in a sample from a subject, where the presence of a polymorphic variant is indicative of a risk of breast cancer.

Thus, featured herein is a method for detecting a subject at risk of breast cancer or the risk of breast cancer in a subject, which comprises detecting the presence or absence of a polymorphic variation associated with breast cancer at a polymorphic site in a nucleotide sequence set forth in SEQ ID NO: 1-5 in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 1-5; (b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1-5; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1-5 or a nucleotide sequence about 90% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1-5; and (d) a fragment of a nucleotide sequence of (a), (b), or (c), often a fragment that includes a polymorphic site associated with breast cancer; whereby the presence of the polymorphic variation is indicative of a risk of breast cancer in the subject. In certain embodiments, determining the presence of a combination of two or more polymorphic variants associated with breast cancer in one or more nucleotide sequences of the sample is determined to identify a subject at risk of breast cancer and/or risk of breast cancer.

A risk of developing aggressive forms of breast cancer likely to metastasize or invade surrounding tissues (e.g., Stage IIIA, IIIB, and IV breast cancers), and subjects at risk of developing aggressive forms of breast cancer also may be identified by the methods described herein. These methods include collecting phenotype information from subjects having breast cancer, which includes the stage of progression of the breast cancer, and performing a secondary phenotype analysis to detect the presence or absence of one or more polymorphic variations associated with a particular stage form of breast cancer. Thus, detecting the presence or absence of one or more polymorphic variations in a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence associated with a late stage form of breast cancer often is prognostic and/or diagnostic of an aggressive form of the cancer.

Results from prognostic tests may be combined with other test results to diagnose breast cancer. For example, prognostic results may be gathered, a patient sample may be ordered based on a determined predisposition to breast cancer, the patient sample is analyzed,-and the results of the analysis may be utilized to diagnose breast cancer. Also breast cancer diagnostic methods can be developed from studies used to generate prognostic/diagnostic methods in which populations are stratified into subpopulations having different progressions of breast cancer. In another embodiment, prognostic results may be gathered; a patient's risk factors for developing breast cancer analyzed (e.g., age, race, family history, age of first menstrual cycle, age at birth of first child); and a patient sample may be ordered based on a determined predisposition to breast cancer. In an alternative embodiment, the results from predisposition analyses described herein may be combined with other test results indicative of breast cancer, which were previously, concurrently, or subsequently gathered with respect to the predisposition testing. In these embodiments, the combination of the prognostic test results with other test results can be probative of breast cancer, and the combination can be utilized as a breast cancer diagnostic. The results of any test indicative of breast cancer known in the art may be combined with the methods described herein. Examples of such tests are mammography (e.g., a more frequent and/or earlier mammography regimen may be prescribed); breast biopsy and optionally a biopsy from another tissue; breast ultrasound and optionally an ultrasound analysis of another tissue; breast magnetic resonance imaging (MRI) and optionally an MRI analysis of another tissue; electrical impedance (T-scan) analysis of breast and optionally of another tissue; ductal lavage; nuclear medicine analysis (e.g., scintimammography); BRCA1 and/or BRCA2 sequence analysis results; and thermal imaging of the breast and optionally of another tissue. Testing may be performed on tissue other than breast to diagnose the occurrence of metastasis (e.g., testing of the lymph node).

Risk of breast cancer sometimes is expressed as a probability, such as an odds ratio, percentage, or risk factor. The risk is based upon the presence or absence of one or more polymorphic variants described herein, and also may be based in part upon phenotypic traits of the individual being tested. Methods for calculating predispositions based upon patient data are well known (see, e.g., Agresti, Categorical Data Analysis, 2nd Ed. 2002. Wiley). Allelotyping and genotyping analyses may be carried out in populations other than those exemplified herein to enhance the predictive power of the prognostic method. These further analyses are executed in view of the exemplified procedures described herein, and may be based upon the same polymorphic variations or additional polymorphic variations. Risk determinations for breast cancer are useful in a variety of applications. In one embodiment, breast cancer risk determinations are used by clinicians to direct appropriate detection, preventative and treatment procedures to subjects who most require these. In another embodiment, breast cancer risk determinations are used by health insurers for preparing actuarial tables and for calculating insurance premiums.

The nucleic acid sample typically is isolated from a biological sample obtained from a subject. For example, nucleic acid can be isolated from blood, saliva, sputum, urine, cell scrapings, and biopsy tissue. The nucleic acid sample can be isolated from a biological sample using standard techniques, such as the technique described in Example 2. As used herein, the term “subject” refers primarily to humans but also refers to other mammals such as dogs, cats, and ungulates (e.g., cattle, sheep, and swine). Subjects also include avians (e.g., chickens and turkeys), reptiles, and fish (e.g., salmon), as embodiments described herein can be adapted to nucleic acid samples isolated from any of these organisms. The nucleic acid sample may be isolated from the subject and then directly utilized in a method for determining the presence of a polymorphic variant, or alternatively, the sample may be isolated and then stored (e.g., frozen) for a period of time before being subjected to analysis.

The presence or absence of a polymorphic variant is determined using one or both chromosomal complements represented in the nucleic acid sample. Determining the presence or absence of a polymorphic variant in both chromosomal complements represented in a nucleic acid sample from a subject having a copy of each chromosome is useful for determining the zygosity of an individual for the polymorphic variant (i.e., whether the individual is homozygous or heterozygous for the polymorphic variant). Any oligonucleotide-based diagnostic may be utilized to determine whether a sample includes the presence or absence of a polymorphic variant in a sample. For example, primer extension methods, ligase sequence determination methods (e.g., U.S. Pat. Nos. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692; 6,110,684; and 6,183,958), microarray sequence determination methods, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism detection (SSCP) (e.g., U.S. Pat. Nos. 5,891,625 and 6,013,499), PCR-based assays (e.g., TAQMAN® PCR System (Applied Biosystems)), and nucleotide sequencing methods may be used.

Oligonucleotide extension methods typically involve providing a pair of oligonucleotide primers in a polymerase chain reaction (PCR) or in other nucleic acid amplification methods for the purpose of amplifying a region from the nucleic acid sample that comprises the polymorphic variation. One oligonucleotide primer is complementary to a region 3′ of the polymorphism and the other is complementary to a region 5′ of the polymorphism. A PCR primer pair may be used in methods disclosed in U.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143; 6,140,054; WO 01/27327; and WO 01/27329 for example. PCR primer pairs may also be used in any commercially available machines that perform PCR, such as any of the GENEAMP® Systems available from Applied Biosystems. Also, those of ordinary skill in the art will be able to design oligonucleotide primers based upon a nucleotide sequence set forth in SEQ ID NO: 1-5 without undue experimentation using knowledge readily available in the art.

Also provided is an extension oligonucleotide that hybridizes to the amplified fragment adjacent to the polymorphic variation. As used herein, the term “adjacent” refers to the 3′ end of the extension oligonucleotide being often 1 nucleotide from the 5′ end of the polymorphic site, and sometimes 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5′ end of the polymorphic site, in the nucleic acid when the extension oligonucleotide is hybridized to the nucleic acid. The extension oligonucleotide then is extended by one or more nucleotides, and the number and/or type of nucleotides that are added to the extension oligonucleotide determine whether the polymorphic variant is present. Oligonucleotide extension methods are disclosed, for example, in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039. Oligonucleotide extension methods using mass spectrometry are described, for example, in U.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242; 5,928,906; 6,043,031; and 6,194,144, and a method often utilized is described herein in Example 2. Multiple extension oligonucleotides may be utilized in one reaction, which is referred to herein as “multiplexing.”

A microarray can be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A microarray may include any oligonucleotides described herein, and methods for making and using oligonucleotide microarrays suitable for diagnostic use are disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330; 5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681; 6,156,501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273; WO 00/52625; WO 01/25485; and WO 01/29259. The microarray typically comprises a solid support and the oligonucleotides may be linked to this solid support by covalent bonds or by non-covalent interactions. The oligonucleotides may also be linked to the solid support directly or by a spacer molecule. A microarray may comprise one or more oligonucleotides complementary to a polymorphic site set forth in SEQ ID NO: 1-5 or below.

A kit also may be utilized for determining whether a polymorphic variant is present or absent in a nucleic acid sample. A kit often comprises one or more pairs of oligonucleotide primers useful for amplifying a fragment of a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence or a substantially identical sequence thereof, where the fragment includes a polymorphic site. The kit sometimes comprises a polymerizing agent, for example, a thermostable nucleic acid polymerase such as one disclosed in U.S. Pat. Nos. 4,889,818 or 6,077,664. Also, the kit often comprises an elongation oligonucleotide that hybridizes to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence in a nucleic acid sample adjacent to the polymorphic site. Where the kit includes an elongation oligonucleotide, it also often comprises chain elongating nucleotides, such as dATP, dTTP, dGTP, dCTP, and dITP, including analogs of dATP, dTTP, dGTP, dCTP and dITP, provided that such analogs are substrates for a thermostable nucleic acid polymerase and can be incorporated into a nucleic acid chain elongated from the extension oligonucleotide. Along with chain elongating nucleotides would be one or more chain terminating nucleotides such as ddATP, ddTTP, ddGTP, ddCTP, and the like. In an embodiment, the kit comprises one or more oligonucleotide primer pairs, a polymerizing agent, chain elongating nucleotides, at least one elongation oligonucleotide, and one or more chain terminating nucleotides. Kits optionally include buffers, vials, microtiter plates, and instructions for use.

An individual identified as being at risk of breast cancer may be heterozygous or homozygous with respect to the allele associated with a higher risk of breast cancer. A subject homozygous for an allele associated with an increased risk of breast cancer is at a comparatively high risk of breast cancer, a subject heterozygous for an allele associated with an increased risk of breast cancer is at a comparatively intermediate risk of breast cancer, and a subject homozygous for an allele associated with a decreased risk of breast cancer is at a comparatively low risk of breast cancer. A genotype may be assessed for a complementary strand, such that the complementary nucleotide at a particular position is detected.

Also featured are methods for determining risk of breast cancer and/or identifying a subject at risk of breast cancer by contacting a polypeptide or protein encoded by a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence from a subject with an antibody that specifically binds to an epitope associated with increased risk of breast cancer in the polypeptide. In certain embodiments, the antibody specifically binds to an epitope that comprises a lysine at amino acid position 237 of SEQ ID NO: 12, a proline at amino acid position 413 of SEQ ID NO: 16 or a glutamine at amino acid position 63 of SEQ ID NO: 16.

Applications of Prognostic and Diagnostic Results to Pharmacogenomic Methods

Pharmacogenomics is a discipline that involves tailoring a treatment for a subject according to the subject's genotype. For example, based upon the outcome of a prognostic test described herein, a clinician or physician may target pertinent information and preventative or therapeutic treatments to a subject who would be benefited by the information or treatment and avoid directing such information and treatments to a subject who would not be benefited (e.g., the treatment has no therapeutic effect and/or the subject experiences adverse side effects). As therapeutic approaches for breast cancer continue to evolve and improve, the goal of treatments for breast cancer related disorders is to intervene even before clinical signs (e.g., identification of lump in the breast) first manifest. Thus, genetic markers associated with susceptibility to breast cancer prove useful for early diagnosis, prevention and treatment of breast cancer.

The following is an example of a pharmacogenomic embodiment. A particular treatment regimen can exert a differential effect depending upon the subject's genotype. Where a candidate therapeutic exhibits a significant interaction with a major allele and a comparatively weak interaction with a minor allele (e.g., an order of magnitude or greater difference in the interaction), such a therapeutic typically would not be administered to a subject genotyped as being homozygous for the minor allele, and sometimes not administered to a subject genotyped as being heterozygous for the minor allele. In another example, where a candidate therapeutic is not significantly toxic when administered to subjects who are homozygous for a major allele but is comparatively toxic when administered to subjects heterozygous or homozygous for a minor allele, the candidate therapeutic is not typically administered to subjects who are genotyped as being heterozygous or homozygous with respect to the minor allele.

The methods described herein are applicable to pharmacogenomic methods for detecting, preventing, alleviating and/or treating breast cancer. For example, a nucleic acid sample from an individual may be subjected to a genetic test described herein. Where one or more polymorphic variations associated with increased risk of breast cancer are identified in a subject, information for detecting, preventing or treating breast cancer and/or one or more breast cancer detection, prevention and/or treatment regimens then may be directed to and/or prescribed to that subject.

In certain embodiments, a detection, prevenative and/or treatment regimen is specifically prescribed and/or administered to individuals who will most benefit from it based upon their risk of developing breast cancer assessed by the methods described herein. Thus, provided are methods for identifying a subject at risk of breast cancer and then prescribing a detection, therapeutic or preventative regimen to individuals identified as being at risk of breast cancer. Thus, certain embodiments are directed to methods for treating breast cancer in a subject, reducing risk of breast cancer in a subject, or early detection of breast cancer in a subject, which comprise: detecting the presence or absence of a polymorphic variant associated with breast cancer in a nucleic acid sample from a subject, where the nucleotide sequence comprises a polynucleotide sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 1-5; (b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1-5; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 1-5 or a nucleotide sequence about 90% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1-5; and (d) a fragment of a nucleotide sequence of (a), (b), or (c), sometimes comprising a polymorphic site associated with breast cancer; and prescribing or administering a breast cancer treatment regimen, preventative regimen and/or detection regimen to a subject from whom the sample originated where the presence of one or more polymorphic variations associated with breast cancer are detected in the nucleotide sequence. In these methods, genetic results may be utilized in combination with other test results to diagnose breast cancer as described above. Other test results include but are not limited to mammography results, imaging results, biopsy results and results from BRCA1 or BRAC2 test results, as described above.

Detection regimens include one or more mammography procedures, a regular mammography regimen (e.g., once a year, or once every six, four, three or two months); an early mammography regimen (e.g., mammography tests are performed beginning at age 25, 30, or 35); one or more biopsy procedures (e.g., a regular biopsy regimen beginning at age 40); breast biopsy and biopsy from other tissue; breast ultrasound and optionally ultrasound analysis of another tissue; breast magnetic resonance imaging (MRI) and optionally MRI analysis of another tissue; electrical impedance (T-scan) analysis of breast and optionally another tissue; ductal lavage; nuclear medicine analysis (e.g., scintimammography); BRCA1 and/or BRCA2 sequence analysis results; and/or thermal imaging of the breast and optionally another tissue.

Treatments sometimes are preventative (e.g., is prescribed or administered to reduce the probability that a breast cancer associated condition arises or progresses), sometimes are therapeutic, and sometimes delay, alleviate or halt the progression of breast cancer. Any known preventative or therapeutic treatment for alleviating or preventing the occurrence of breast cancer is prescribed and/or administered. For example, certain preventative treatments often are prescribed to subjects having a predisposition to breast cancer and where the subject is not diagnosed with breast cancer or is diagnosed as having symptoms indicative of early stage breast cancer (e.g., stage 1). For subjects not diagnosed as having breast cancer, any preventative treatments known in the art can be prescribed and administered, which include selective hormone receptor modulators (e.g., selective estrogen receptor modulators (SERMs) such as tamoxifen, reloxifene, and toremifene); compositions that prevent production of hormones (e.g., aramotase inhibitors that prevent the production of estrogen in the adrenal gland, such as exemestane, letrozole, anastrozol, groserelin, and megestrol); other hormonal treatments (e.g., goserelin acetate and fulvestrant); biologic response modifiers such as antibodies (e.g., trastuzumab (herceptin/HER2)); surgery (e.g., lumpectomy and mastectomy); drugs that delay or halt metastasis (e.g., pamidronate disodium); and alternative/complementary medicine (e.g., acupuncture, acupressure, moxibustion, qi gong, reiki, ayurveda, vitamins, minerals, and herbs (e.g., astragalus root, burdock root, garlic, green tea, and licorice root)).

The use of breast cancer treatments are well known in the art, and include surgery, chemotherapy and/or radiation therapy. Any of the treatments may be used in combination to treat or prevent breast cancer (e.g., surgery followed by radiation therapy or chemotherapy). Examples of chemotherapy combinations used to treat breast cancer include: cyclophosphamide (Cytoxan), methotrexate (Amethopterin, Mexate, Folex), and fluorouracil (Fluorouracil, 5-Fu, Adrucil), which is referred to as CMF; cyclophosphamide, doxorubicin (Adriamycin), and fluorouracil, which is referred to as CAF; and doxorubicin (Adriamycin) and cyclophosphamide, which is referred to as AC.

As breast cancer preventative and treatment information can be specifically targeted to subjects in need thereof (e.g., those at risk of developing breast cancer or those that have early signs of breast cancer), provided herein is a method for preventing or reducing the risk of developing breast cancer in a subject, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with breast cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying a subject with a predisposition to breast cancer, whereby the presence of the polymorphic variation is indicative of a predisposition to breast cancer in the subject; and (c) if such a predisposition is identified, providing the subject with information about methods or products to prevent or reduce breast cancer or to delay the onset of breast cancer. Also provided is a method of targeting information or advertising to a subpopulation of a human population based on the subpopulation being genetically predisposed to a disease or condition, which comprises: (a) detecting the presence or absence of a polymorphic variation associated with breast cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) identifying the subpopulation of subjects in which the polymorphic variation is associated with breast cancer; and (c) providing information only to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition.

Pharmacogenomics methods also may be used to analyze and predict a response to a breast cancer treatment or a drug. For example, if pharmacogenomics analysis indicates a likelihood that an individual will respond positively to a breast cancer treatment with a particular drug, the drug may be administered to the individual. Conversely, if the analysis indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects. The response to a therapeutic treatment can be predicted in a background study in which subjects in any of the following populations are genotyped: a population that responds favorably to a treatment regimen, a population that does not respond significantly to a treatment regimen, and a population that responds adversely to a treatment regiment (e.g., exhibits one or more side effects). These populations are provided as examples and other populations and subpopulations may be analyzed. Based upon the results of these analyses, a subject is genotyped to predict whether he or she will respond favorably to a treatment regimen, not respond significantly to a treatment regimen, or respond adversely to a treatment regimen.

The methods described herein also are applicable to clinical drug trials. One or more polymorphic variants indicative of response to an agent for treating breast cancer or to side effects to an agent for treating breast cancer may be identified using the methods described herein. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems. In certain embodiments, the agent for treating breast cancer described herein targets GP6, LAMA4, CHGB, LOC338749 or TTN or a target in the GP6, LAMA4, CHGB, LOC338749 or TTN pathway.

Thus, another embodiment is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: (a) obtaining a nucleic acid sample from an individual; (b) determining the identity of a polymorphic variation which is associated with a positive response to the treatment or the drug, or at least one polymorphic variation which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and (c) including the individual in the clinical trial if the nucleic acid sample contains said polymorphic variation associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said polymorphic variation associated with a negative response to the treatment or the drug. In addition, the methods for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination. The polymorphic variation may be in a sequence selected individually or in any combination from the group consisting of (i) a polynucleotide sequence set forth in SEQ ID NO: 1-5; (ii) a polynucleotide sequence that is 90% or more identical to a nucleotide sequence set forth in SEQ ID NO: 1-5; (iii) a polynucleotide sequence that encodes a polypeptide having an amino acid sequence identical to or 90% or more identical to an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO: 1-5; and (iv) a fragment of a polynucleotide sequence of (i), (ii), or (iii) comprising the polymorphic site. The including step (c) optionally comprises administering the drug or the treatment to the individual if the nucleic acid sample contains the polymorphic variation associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.

Also provided herein is a method of partnering between a diagnostic/prognostic testing provider and a provider of a consumable product, which comprises: (a) the diagnostic/prognostic testing provider detects the presence or absence of a polymorphic variation associated with breast cancer at a polymorphic site in a nucleotide sequence in a nucleic acid sample from a subject; (b) the diagnostic/prognostic testing provider identifies the subpopulation of subjects in which the polymorphic variation is associated with breast cancer; (c) the diagnostic/prognostic testing provider forwards information to the subpopulation of subjects about a particular product which may be obtained and consumed or applied by the subject to help prevent or delay onset of the disease or condition; and (d) the provider of a consumable product forwards to the diagnostic test provider a fee every time the diagnostic/prognostic test provider forwards information to the subject as set forth in step (c) above.

Compositions Comprising Breast Cancer-Directed Molecules

Featured herein is a composition comprising a breast cancer cell and one or more molecules specifically directed and targeted to a nucleic acid comprising a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence or a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. Such directed molecules include, but are not limited to, a compound that binds to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid or a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide; a RNAi or siRNA molecule having a strand complementary to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence; an antisense nucleic acid complementary to an RNA encoded by a GP6, LAMA4, CHGB, LOC338749 or TTN DNA sequence; a ribozyme that hybridizes to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence; a nucleic acid aptamer that specifically binds a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide; and an antibody that specifically binds to a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or binds to a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid. In certain embodiments, the antibody specifically binds to an epitope that comprises a a lysine at amino acid position 237 of SEQ ID NO: 12, a proline at amino acid position 413 of SEQ ID NO: 16 or a glutamine at amino acid position 63 of SEQ ID NO: 16. In specific embodiments, the breast cancer directed molecule interacts with a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid or polypeptide variant associated with breast cancer. In other embodiments, the breast cancer directed molecule interacts with a polypeptide involved in the GP6, LAMA4, CHGB, LOC338749 or TTN signal pathway, or a nucleic acid encoding such a polypeptide. Polypeptides involved in the GP6, LAMA4, CHGB, LOC338749 or TTN signal pathway are discussed herein.

Compositions sometimes include an adjuvant known to stimulate an immune response, and in certain embodiments, an adjuvant that stimulates a T-cell lymphocyte response. Adjuvants are known, including but not limited to an aluminum adjuvant (e.g., aluminum hydroxide); a cytokine adjuvant or adjuvant that stimulates a cytokine response (e.g., interleukin (IL)-12 and/or γ-interferon cytokines); a Freund-type mineral oil adjuvant emulsion (e.g., Freund's complete or incomplete adjuvant); a synthetic lipoid compound; a copolymer adjuvant (e.g., TitreMax); a saponin; Quil A; a liposome; an oil-in-water emulsion (e.g., an emulsion stabilized by Tween 80 and pluronic polyoxyethlene/polyoxypropylene block copolymer (Syntex Adjuvant Formulation); TitreMax; detoxified endotoxin (MPL) and mycobacterial cell wall components (TDW, CWS) in 2% squalene (Ribi Adjuvant System)); a muramyl dipeptide; an immune-stimulating complex (ISCOM, e.g., an Ag-modified saponin/cholesterol micelle that forms stable cage-like structure); an aqueous phase adjuvant that does not have a depot effect (e.g., Gerbu adjuvant); a carbohydrate polymer (e.g., AdjuPrime); L-tyrosine; a manide-oleate compound (e.g., Montanide); an ethylene-vinyl acetate copolymer (e.g., Elvax 40W1,2); or lipid A, for example. Such compositions are useful for generating an immune response against a breast cancer directed molecule (e.g., an HLA-binding subsequence within a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 1-5). In such methods, a peptide having an amino acid subsequence of a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 1-5 is delivered to a subject, where the subsequence binds to an HLA molecule and induces a CTL lymphocyte response. The peptide sometimes is delivered to the subject as an isolated peptide or as a minigene in a plasmid that encodes the peptide. Methods for identifying HLA-binding subsequences in such polypeptides are known (see e.g., publication WO02/20616 and PCT application US98/01373 for methods of identifying such sequences).

The breast cancer cell may be in a group of breast cancer cells and/or other types of cells cultured in vitro or in a tissue having breast cancer cells (e.g., a melanocytic lesion) maintained in vitro or present in an animal in vivo (e.g., a rat, mouse, ape or human). In certain embodiments, a composition comprises a component from a breast cancer cell or from a subject having a breast cancer cell instead of the breast cancer cell or in addition to the breast cancer cell, where the component sometimes is a nucleic acid molecule (e.g., genomic DNA), a protein mixture or isolated protein, for example. The aforementioned compositions have utility in diagnostic, prognostic and pharmacogenomic methods described previously and in breast cancer therapeutics described hereafter. Certain breast cancer molecules are described in greater detail below.

Compounds

Compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive (see, e.g., Zuckermann et al., J. Med. Chem.37: 2678-85 (1994)); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; “one-bead one-compound” library methods; and synthetic library methods using affinity chromatography selection. Biological library and peptoid library approaches are typically limited to peptide libraries, while the other approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, (1997)). Examples of methods for synthesizing molecular libraries are described, for example, in DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90: 6909 (1993); Erb et al., Proc. Natl. Acad. Sci. USA 91: 11422 (1994); Zuckermann et al., J. Med. Chem. 37: 2678 (1994); Cho et al, Science 261: 1303 (1993); Carrell et al., Angew. Chem. Int. Ed. Engl. 33: 2059 (1994); Carell et al., Angew. Chem. Int. Ed. Engl. 33: 2061 (1994); and in Gallop et al., J. Med. Chem. 37: 1233 (1994).

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13: 412-421 (1992)), or on beads (Lam, Nature 354: 82-84 (1991)), chips (Fodor, Nature 364: 555-556 (1993)), bacteria or spores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869 (1992)) or on phage (Scott and Smith, Science 249: 386-390 (1990); Devlin, Science 249: 404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. 87: 6378-6382 (1990); Felici, J. Mol. Biol. 222: 301-310 (1991); Ladner supra.).

A compound sometimes alters expression and sometimes alters activity of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide and may be a small molecule. Small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Antisense Nucleic Acid Molecules, Ribozymes, RNAi, siRNA and Modified Nucleic Acid Molecules

An “antisense” nucleic acid refers to a nucleotide sequence complementary to a “sense” nucleic acid encoding a polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire coding strand in SEQ ID NO: 1-11, or to a portion thereof or a substantially identical sequence thereof. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence in SEQ ID NO: 1-11 (e.g., 5′ and 3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementary to the entire coding region of an mRNA encoded by a nucleotide sequence in SEQ ID NO: 1-5 (e.g., SEQ ID NO: 6-11), and often the antisense nucleic acid is an oligonucleotide antisense to only a portion of a coding or noncoding region of the mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the mRNA, e.g., between the −10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. The antisense nucleic acids, which include the ribozymes described hereafter, can be designed to target a nucleotide sequence in SEQ ID NO: 1-11, often a variant associated with breast cancer, or a substantially identical sequence thereof. Among the variants, minor alleles and major alleles can be targeted, and those associated with a higher risk of breast cancer are often designed, tested, and administered to subjects.

An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using standard procedures. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

When utilized as therapeutics, antisense nucleic acids typically are administered to a subject (e.g., by direct injection at a tissue site) or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide and thereby inhibit expression of the polypeptide, for example, by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then are administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, for example, by linking antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules are achieved by incorporating a strong promoter, such as a pol II or pol III promoter, in the vector construct.

Antisense nucleic acid molecules sometimes are *-anomeric nucleic acid molecules. An *-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual *-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15: 6625-6641 (1987)). Antisense nucleic acid molecules can also comprise a 2′-o-methylribonucleotide (Inoue et al., Nucleic Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215: 327-330 (1987)). Antisense nucleic acids sometimes are composed of DNA or PNA or any other nucleic acid derivatives described previously.

In another embodiment, an antisense nucleic acid is a ribozyme. A ribozyme having specificity for a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence can include one or more sequences complementary to such a nucleotide sequence, and a sequence having a known catalytic region responsible for mRNA cleavage (see e.g., U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, Nature 334: 585-591 (1988)). For example, a derivative of a Tetrahymena L-19 IVS RNA is sometimes utilized in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA (see e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Also, target mRNA sequences can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see e.g., Bartel & Szostak, Science 261: 1411-1418 (1993)).

Breast cancer directed molecules include in certain embodiments nucleic acids that can form triple helix structures with a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence or a substantially identical sequence thereof, especially one that includes a regulatory region that controls expression of a polypeptide. Gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence or a substantially identical sequence (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of a gene in target cells (see e.g., Helene, Anticancer Drug Des. 6(6): 569-84 (1991); Helene et al., Ann. N.Y. Acad. Sci. 660: 27-36 (1992); and Maher, Bioassays 14(12): 807-15 (1992). Potential sequences that can be targeted for triple helix formation can be increased by creating a so-called “switchback” nucleic acid molecule. Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′ manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

Breast cancer directed molecules include RNAi and siRNA nucleic acids. Gene expression may be inhibited by the introduction of double-stranded RNA (dsRNA), which induces potent and specific gene silencing, a phenomenon called RNA interference or RNAi. See, e.g., Fire et al., U.S. Pat. No. 6,506,559; Tuschl et al. PCT International Publication No. WO 01/75164; Kay et al. PCT International Publication No. WO 03/010180A 1; or Bosher J M, Labouesse, Nat Cell Biol February 2000;2(2):E31-6. This process has been improved by decreasing the size of the double-stranded RNA to 20-24 base pairs (to create small-interfering RNAs or siRNAs) that “switched off” genes in mammalian cells without initiating an acute phase response, i.e., a host defense mechanism that often results in cell death (see, e.g., Caplen et al. Proc Natl Acad Sci U S A. Aug. 14, 2001;98(17):9742-7 and Elbashir et al. Methods February 2002;26(2):199-213). There is increasing evidence of post-transcriptional gene silencing by RNA interference (RNAi) for inhibiting targeted expression in mammalian cells at the mRNA level, in human cells. There is additional evidence of effective methods for inhibiting the proliferation and migration of tumor cells in human patients, and for inhibiting metastatic cancer development (see, e.g., U.S. Patent Application No. US2001000993183; Caplen et al. Proc Natl Acad Sci USA; and Abderrahmani et al. Mol Cell Biol Nov. 21, 2001(21):7256-67).

An “siRNA” or “RNAi” refers to a nucleic acid that forms a double stranded RNA and has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is delivered to or expressed in the same cell as the gene or target gene. “siRNA” refers to short double-stranded RNA formed by the complementary strands. Complementary portions of the siRNA that hybridize to form the double stranded molecule often have substantial or complete identity to the target molecule sequence. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA.

When designing the siRNA molecules, the targeted region often is selected from a given DNA sequence beginning 50 to 100 nucleotides downstream of the start codon. See, e.g., Elbashir et al,. Methods 26:199-213 (2002). Initially, 5′ or 3′ UTRs and regions nearby the start codon were avoided assuming that UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP or RISC endonuclease complex. Sometimes regions of the target 23 nucleotides in length conforming to the sequence motif AA(N19)TT (N, an nucleotide), and regions with approximately 30% to 70% G/C-content (often about 50% G/C-content) often are selected. If no suitable sequences are found, the search often is extended using the motif NA(N21). The sequence of the sense siRNA sometimes corresponds to (N19) TT or N21 (position 3 to 23 of the 23-nt motif), respectively. In the latter case, the 3′ end of the sense siRNA often is converted to TT. The rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense 3′ overhangs. The antisense siRNA is synthesized as the complement to position 1 to 21 of the 23-nt motif. Because position 1 of the 23-nt motif is not recognized sequence-specifically by the antisense siRNA, the 3′-most nucleotide residue of the antisense siRNA can be chosen deliberately. However, the penultimate nucleotide of the antisense siRNA (complementary to position 2 of the 23-nt motif) often is complementary to the targeted sequence. For simplifying chemical synthesis, Tr often is utilized. siRNAs corresponding to the target motifNAR(N17)YNN, where R is purine (A,G) and Y is pyrimidine (C,U), often are selected. Respective 21 nucleotide sense and antisense siRNAs often begin with a purine nucleotide and can also be expressed from pol II expression vectors without a change in targeting site. Expression of RNAs from pol III promoters often is efficient when the first transcribed nucleotide is a purine.

The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Often, the siRNA is about 15 to about 50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, sometimes about 20-30 nucleotides in length or about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The siRNA sometimes is about 21 nucleotides in length. Methods of using siRNA are well known in the art, and specific siRNA molecules may be purchased from a number of companies including Dharmacon Research, Inc.

Antisense, ribozyme, RNAi and siRNA nucleic acids can be altered to form modified nucleic acid molecules. The nucleic acids can be altered at base moieties, sugar moieties or phosphate backbone moieties to improve stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup et al., Bioorganic & Medicinal Chemistry 4 (1): 5-23 (1996)). As used herein, the terms “peptide nucleic acid” or “PNA” refers to a nucleic acid mimic such as a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described, for example, in Hyrup et al., (1996) supra and Perry-O'Keefe et al., Proc. Natl. Acad. Sci. 93: 14670-675 (1996).

PNA nucleic acids can be used in prognostic, diagnostic, and therapeutic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNA nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as “artificial restriction enzymes” when used in combination with other enzymes, (e.g., SI nucleases (Hyrup (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup et al., (1996) supra; Perry-O'Keefe supra).

In other embodiments, oligonucleotides may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across cell membranes (see e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization- triggered cleavage agents (See, e.g., Krol et al., Bio-Techniques 6: 958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5: 539-549 (1988) ). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

Also included herein are molecular beacon oligonucleotide primer and probe molecules having one or more regions complementary to a nucleotide sequence of SEQ ID NO: 1-11 or a substantially identical sequence thereof, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantifying the presence of the nucleic acid in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al., U.S. Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak et al., U.S. Pat. No. 5,876,930.

Antibodies

The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. An antibody sometimes is a polyclonal, monoclonal, recombinant (e.g., a chimeric or humanized), filly human, non-human (e.g., murine), or a single chain antibody. An antibody may have effector function and can fix complement, and is sometimes coupled to a toxin or imaging agent.

A full-length polypeptide or antigenic peptide fragment encoded by a GP6, LAMA4, CHGB, LOC338749 or TTN nucleotide sequence can be used as an immunogen or can be used to identify antibodies made with other immunogens, e.g., cells, membrane preparations, and the like. An antigenic peptide often includes at least 8 amino acid residues of the amino acid sequences encoded by a nucleotide sequence of SEQ ID NO: 1-11, or substantially identical sequence thereof, and encompasses an epitope. Antigenic peptides sometimes include 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, or 30 or more amino acids. Hydrophilic and hydrophobic fragments of polypeptides sometimes are used as immunogens.

Epitopes encompassed by the antigenic peptide are regions located on the surface of the polypeptide (e.g., hydrophilic regions) as well as regions with high antigenicity. For example, an Emini surface probability analysis of the human polypeptide sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface of the polypeptide and are thus likely to constitute surface residues useful for targeting antibody production. The antibody may bind an epitope on any domain or region on polypeptides described herein.

Also, chimeric, humanized, and completely human antibodies are useful for applications which include repeated administration to subjects. Chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al International Application No. PCT/US86/02269; Akira, et al European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al European Patent Application 173,494; Neuberger et al PCT International Publication No. WO 86/01533; Cabilly et al U.S. Pat. No. 4,816,567; Cabilly et al European Patent Application 125,023; Better et al., Science 240: 1041-1043 (1988); Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443 (1987); Liu et al., J. Immunol. 139: 3521-3526 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218 (1987); Nishimura et al., Canc. Res. 47: 999-1005 (1987); Wood et al., Nature 314: 446-449 (1985); and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559 (1988); Morrison, S. L., Science 229:1202-1207 (1985); Oi et al., BioTechniques 4: 214 (1986); Winter U.S. Pat. No. 5,225,539; Jones et al., Nature 321: 552-525 (1986); Verhoeyan et al., Science 239: 1534; and Beidler et al., J. Immunol. 141: 4053-4060 (1988).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. See, for example, Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995); and U.S. Pat. Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, Calif.) and Medarex, Inc. (Princeton, N.J.), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. Completely human antibodies that recognize a selected epitope also can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody (e.g., a murine antibody) is used to guide the selection of a completely human antibody recognizing the same epitope. This technology is described for example by Jespers et al., Bio/Technology 12: 899-903 (1994).

Antibody can be a single chain antibody. A single chain antibody (scFV) can be engineered (see, e.g., Colcher et al., Ann. NY Acad. Sci. 880: 263-80 (1999); and Reiter, Clin. Cancer Res. 2: 245-52 (1996)). Single chain antibodies can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target polypeptide.

Antibodies also may be selected or modified so that they exhibit reduced or no ability to bind an Fc receptor. For example, an antibody may be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor (e.g., it has a mutagenized or deleted Fc receptor binding region).

Also, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

Antibody conjugates can be used for modifying a given biological response. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Also, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, for example.

An antibody (e.g., monoclonal antibody) can be used to isolate target polypeptides by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an antibody can be used to detect a target polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H. Also, an antibody can be utilized as a test molecule for determining whether it can treat breast cancer, and as a therapeutic for administration to a subject for treating breast cancer.

An antibody can be made by immunizing with a purified antigen, or a fragment thereof, e.g., a fragment described herein, a membrane associated antigen, tissues, e.g., crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions.

Included herein are antibodies which bind only a native polypeptide, only denatured or otherwise non-native polypeptide, or which bind both, as well as those having linear or conformational epitopes. Conformational epitopes sometimes can be identified by selecting antibodies that bind to native but not denatured polypeptide. Also featured are antibodies that specifically bind to a polypeptide variant associated with breast cancer.

Screening Assays

Featured herein are methods for identifying a candidate therapeutic for treating breast cancer. The methods comprise contacting a test molecule with a target molecule in a system. A “target molecule” as used herein refers to a nucleic acid of SEQ ID NO: 1-11, a substantially identical nucleic acid thereof, or a fragment thereof, and an encoded polypeptide of the foregoing. The method also comprises determining the presence or absence of an interaction between the test molecule and the target molecule, where the presence of an interaction between the test molecule and the nucleic acid or polypeptide identifies the test molecule as a candidate breast cancer therapeutic. The interaction between the test molecule and the target molecule may be quantified.

Test molecules and candidate therapeutics include, but are not limited to, compounds, anti sense nucleic acids, siRNA molecules, ribozymes, polypeptides or proteins encoded by a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acids, or a substantially identical sequence or fragment thereof, and immunotherapeutics (e.g., antibodies and HLA-presented polypeptide fragments). A test molecule or candidate therapeutic may act as a modulator of target molecule concentration or target molecule function in a system. A “modulator” may agonize (i.e., up-regulates) or antagonize (i.e., down-regulates) a target molecule concentration partially or completely in a system by affecting such cellular functions as DNA replication and/or DNA processing (e.g., DNA methylation or DNA repair), RNA transcription and/or RNA processing (e.g., removal of intronic sequences and/or translocation of spliced mRNA from the nucleus), polypeptide production (e.g., translation of the polypeptide from mRNA), and/or polypeptide post-translational modification (e.g., glycosylation, phosphorylation, and proteolysis of pro-polypeptides). A modulator may also agonize or antagonize a biological function of a target molecule partially or completely, where the function may include adopting a certain structural conformation, interacting with one or more binding partners, ligand binding, catalysis (e.g., phosphorylation, dephosphorylation, hydrolysis, methylation, and isomerization), and an effect upon a cellular event (e.g., effecting progression of breast cancer).

As used herein, the term “system” refers to a cell free in vitro environment and a cell-based environment such as a collection of cells, a tissue, an organ, or an organism. A system is “contacted” with a test molecule in a variety of manners, including adding molecules in solution and allowing them to interact with one another by diffusion, cell injection, and any administration routes in an animal. As used herein, the term “interaction” refers to an effect of a test molecule on test molecule, where the effect sometimes is binding between the test molecule and the target molecule, and sometimes is an observable change in cells, tissue, or organism.

There are many standard methods for detecting the presence or absence of an interaction between a test molecule and a target molecule. For example, titrametric, acidimetric, radiometric, NMR, monolayer, polarographic, spectrophotometric, fluorescent, and ESR assays probative of a target molecule interaction may be utilized.

In general, an interaction can be determined by labeling the test molecule and/or the GP6, LAMA4, CHGB, LOC338749 or TTN molecule, where the label is covalently or non-covalently attached to the test molecule or GP6, LAMA4, CHGB, LOC338749 or TTN molecule. The label is sometimes a radioactive molecule such as ¹²⁵I, ¹³¹I, ³⁵S or ³H, which can be detected by direct counting of radioemission or by scintillation counting. Also, enzymatic labels such as horseradish peroxidase, alkaline phosphatase, or luciferase may be utilized where the enzymatic label can be detected by determining conversion of an appropriate substrate to product. Also, presence or absence of an interaction can be determined without labeling. For example, a microphysiometer (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indication of an interaction between a test molecule and GP6, LAMA4, CHGB, LOC338749 or TTN (McConnell, H. M. et al., Science 257: 1906-1912 (1992)).

In cell-based systems, cells typically include a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid or polypeptide or variants thereof and are often of mammalian origin, although the cell can be of any origin. Whole cells, cell homogenates, and cell fractions (e.g., cell membrane fractions) can be subjected to analysis. Where interactions between a test molecule with a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or variant thereof are monitored, soluble and/or membrane bound forms of the polypeptide or variant may be utilized. Where membrane-bound forms of the polypeptide are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

An interaction between two molecules also can be detected by monitoring fluorescence energy transfer (FET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al. U.S. Pat. No. 4,868,103). A fluorophore label on a first, “donor” molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, “acceptor” molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the “donor” polypeptide molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the “acceptor” molecule label may be differentiated from that of the “donor”. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the “acceptor” molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the presence or absence of an interaction between a test molecule and a GP6, LAMA4, CHGB, LOC338749 or TTN molecule can be effected by using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander & Urbaniczk, Anal. Chem. 63: 2338-2345 (1991) and Szabo et al., Curr. Opin. Struct. Biol. 5: 699-705 (1995)). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

In another embodiment, the GP6, LAMA4, CHGB, LOC338749 or TTN molecule or test molecules are anchored to a solid phase. The GP6, LAMA4, CHGB, LOC338749 or TTN molecule/test molecule complexes anchored to the solid phase can be detected at the end of the reaction. The target GP6, LAMA4, CHGB, LOC338749 or TTN molecule is often anchored to a solid surface, and the test molecule, which is not anchored, can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize a GP6, LAMA4, CHGB, LOC338749 or TTN molecule, an anti-GP6, LAMA4, CHGB, LOC338749 or TTN antibody, or test molecules to facilitate separation of complexed from uncomplexed forms of GP6, LAMA4, CHGB, LOC338749 or TTN molecules and test molecules, as well as to accommodate automation of the assay. Binding of a test molecule to a GP6, LAMA4, CHGB, LOC338749 or TTN molecule can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion polypeptide can be provided which adds a domain that allows a GP6, LAMA4, CHGB, LOC338749 or TTN molecule to be bound to a matrix. For example, glutathione-S-transferase/GP6, LAMA4, CHGB, LOC338749 or TTN fusion polypeptides or glutathione-S-transferase/target fusion polypeptides can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivitized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target polypeptide or GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of GP6, LAMA4, CHGB, LOC338749 or TTN binding or activity determined using standard techniques.

Other techniques for immobilizing a GP6, LAMA4, CHGB, LOC338749 or TTN molecule on matrices include using biotin and streptavidin. For example, biotinylated GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactive with GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or test molecules but which do not interfere with binding of the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide to its test molecule. Such antibodies can be derivitized to the wells of the plate, and unbound target or GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or test molecule.

Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G., and Minton, A. P., Trends Biochem Sci August;18(8): 284-7 (1993)); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology, J. Wiley: New York (1999)); and immunoprecipitation (see, for example, Ausubel, F. et al., eds. Current Protocols in Molecular Biology, J. Wiley: New York (1999)). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, J Mol. Recognit. Winter; 11(1-6): 141-8 (1998); Hage & Tweed, J. Chromatogr. B Biomed. Sci. Appl. October 10; 699 (1-2): 499-525 (1997)). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

In another embodiment, modulators of GP6, LAMA4, CHGB, LOC338749 or TTN expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of GP6, LAMA4, CHGB, LOC338749 or TTN mRNA or polypeptide evaluated relative to the level of expression of GP6, LAMA4, CHGB, LOC338749 or TTN mRNA or polypeptide in the absence of the candidate compound. When expression of GP6, LAMA4, CHGB, LOC338749 or TTN mRNA or polypeptide is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GP6, LAMA4, CHGB, LOC338749 or TTN mRNA or polypeptide expression. Alternatively, when expression of GP6, LAMA4, CHGB, LOC338749 or TTN mRNA or polypeptide is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GP6, LAMA4, CHGB, LOC338749 or TTN mRNA or polypeptide expression. The level of GP6, LAMA4, CHGB, LOC338749 or TTN mRNA or polypeptide expression can be determined by methods described herein for detecting GP6, LAMA4, CHGB, LOC338749 or TTN mRNA or polypeptide.

In another embodiment, binding partners that interact with a GP6, LAMA4, CHGB, LOC338749 or TTN molecule are detected. The GP6, LAMA4, CHGB, LOC338749 or TTN molecules can interact with one or more cellular or extracellular macromolecules, such as polypeptides, in vivo, and these molecules that interact with GP6, LAMA4, CHGB, LOC338749 or TTN molecules are referred to herein as “binding partners.” Molecules that disrupt such interactions can be useful in regulating the activity of the target gene product. Such molecules can include, but are not limited to molecules such as antibodies, peptides, and small molecules. Target genes/products for use in this embodiment often are the GP6, LAMA4, CHGB, LOC338749 or TTN genes herein identified. In an alternative embodiment, provided is a method for determining the ability of the test compound to modulate the activity of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide through modulation of the activity of a downstream effector of a GP6, LAMA4, CHGB, LOC338749 or TTN target molecule. For example, the activity of the effector molecule on an appropriate target can be determined, or the binding of the effector to an appropriate target can be determined, as previously described.

To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), e.g., a substrate, a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence of the test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases where it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.

In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g., a microtiter plate), while the non- anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

In an alternate embodiment, a homogeneous assay can be used. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

Also, binding partners of GP6, LAMA4, CHGB, LOC338749 or TTN molecules can be identified in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 (1993); Madura et al., J. Biol. Chem. 268: 12046-12054 (1993); Bartel et al., Biotechniques 14: 920-924 (1993); Iwabuchi et al., Oncogene 8: 1693-1696 (1993); and Brent WO94/10300), to identify other polypeptides, which bind to or interact with GP6, LAMA4, CHGB, LOC338749 or TTN(“GP6, LAMA4, CHGB, LOC338749 or TTN-binding polypeptides” or “GP6, LAMA4, CHGB, LOC338749 or TTN-bp”) and are involved in GP6, LAMA4, CHGB, LOC338749 or TTN activity. Such GP6, LAMA4, CHGB, LOC338749 or TTN-bps can be activators or inhibitors of signals by the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptides or GP6, LAMA4, CHGB, LOC338749 or TTN targets as, for example, downstream elements of a GP6, LAMA4, CHGB, LOC338749 or TTN-mediated signaling pathway.

A two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified polypeptide (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. (Alternatively the: GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide can be the fused to the activator domain.) If the “bait” and the “prey” polypeptides are able to interact, in vivo, forming a GP6, LAMA4, CHGB, LOC338749 or TTN-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the polypeptide which interacts with the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide.

Candidate therapeutics for treating breast cancer are identified from a group of test molecules that interact with a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid or polypeptide. Test molecules are normally ranked according to the degree with which they interact or modulate (e.g., agonize or antagonize) DNA replication and/or processing, RNA transcription and/or processing, polypeptide production and/or processing, and/or function of GP6, LAMA4, CHGB, LOC338749 or TTN molecules, for example, and then top ranking modulators are selected. In a preferred embodiment, the candidate therapeutic (i.e., test molecule) acts as a GP6, LAMA4, CHGB, LOC338749 or TTN antagonist. Also, pharmacogenomic information described herein can determine the rank of a modulator. Candidate therapeutics typically are formulated for administration to a subject.

Therapeutic Treatments

Formulations or pharmaceutical compositions typically include in combination with a pharmaceutically acceptable carrier, a compound, an antisense nucleic acid, a ribozyme, an antibody, a binding partner that interacts with a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide, a GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid, or a fragment thereof. The formulated molecule may be one that is identified by a screening method described above. Also, formulations may comprise a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or fragment thereof. As used herein, the term “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride sometimes are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation often utilized are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. Molecules can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, active molecules are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/D₅₀. Molecules which exhibit high therapeutic indices often are utilized. While molecules that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such molecules often lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any molecules used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, sometimes about 0.01 to 25 mg/kg body weight, often about 0. I to 20 mg/kg body weight, and more often about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, sometimes between 2 to 8 weeks, often between about 3 to 7 weeks, and more often for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment, or sometimes can include a series of treatments.

With regard to polypeptide formulations, featured herein is a method for treating breast cancer in a subject, which comprises contacting one or more cells in the subject with a first GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide, where the subject comprises a second GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide having one or more polymorphic variations associated with cancer, and where the first polypeptide comprises fewer polymorphic variations associated with cancer than the second polypeptide. The first and second polypeptides are encoded by a nucleic acid which comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1-11; a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-11; a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-11 and a nucleotide sequence 90% or more identical to a nucleotide sequence of SEQ ID NO: 1-11. The subject is often a human.

For antibodies, a dosage of 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg) is often utilized. If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is often appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al., J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193 (1997).

Antibody conjugates can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, .alpha.-interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

For compounds, exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sample weight, for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid described herein, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid molecules can be inserted into vectors and used in gene therapy methods for treating breast cancer. Featured herein is a method for treating breast cancer in a subject, which comprises contacting one or more cells in the subject with a first GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid, where genomic DNA in the subject comprises a second GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid comprising one or more polymorphic variations associated with breast cancer, and where the first GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid comprises fewer polymorphic variations associated with breast cancer. The first and second nucleic acids typically comprise a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1-11; a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-11; a nucleotide sequence that is 90% or more identical to the nucleotide sequence of SEQ ID NO: 1-11, and a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence of SEQ ID NO: 1-11. The subject often is a human.

Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al., (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). Pharmaceutical preparations of gene therapy vectors can include a gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells (e.g., retroviral vectors) the pharmaceutical preparation can include one or more cells which produce the gene delivery system. Examples of gene delivery vectors are described herein.

Pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Pharmaceutical compositions of active ingredients can be administered by any of the paths described herein for therapeutic and prophylactic methods for treating breast cancer. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from pharmacogenomic analyses described herein. As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the GP6, LAMA4, CHGB, LOC338749 or TTN aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of GP6, LAMA4, CHGB, LOC338749 or TTN aberrance, for example, a GP6, LAMA4, CHGB, LOC338749 or TTN molecule, GP6, LAMA4, CHGB, LOC338749 or TTN agonist, or GP6, LAMA4, CHGB, LOC338749 or TTN antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

As discussed, successful treatment of GP6, LAMA4, CHGB, LOC338749 or TTN disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds (e.g., an agent identified using an assays described above) that exhibit negative modulatory activity can be used to prevent and/or treat breast cancer. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)2 and FAb expression library fragments, scFV molecules, and epitope- binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of the target gene can also be used to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances where the target gene encodes an extracellular polypeptide, normal target gene polypeptide often is co-administered into the cell or tissue to maintain the requisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by GP6, LAMA4, CHGB, LOC338749 or TTN expression is through the use of aptamer molecules specific for GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to polypeptide ligands (see, e.g., Osborne, et al., Curr. Opin. Chem. Biol. 1(1): 5-9 (1997); and Patel, D. J., Curr. Opin. Chem. Biol. June;l(l): 32-46 (1997)). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic polypeptide molecules may be, aptamers offer a method by which GP6, LAMA4A, CHGB, LOC338749 or TTN polypeptide activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of GP6, LAMA4, CHGB, LOC338749 or TTN disorders. For a description of antibodies, see the Antibody section above.

In circumstances where injection of an animal or a human subject with a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against GP6, LAMA4, CHGB, LOC338749 or TTN through the use of anti-idiotypic antibodies (see, for example, Herlyn, D., Ann. Med.;31(1): 66-78 (1999); and Bhattacharya-Chatterjee & Foon, Cancer Treat. Res.; 94: 51-68 (1998)). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide. Vaccines directed to a disease characterized by GP6, LAMA4, CHGB, LOC338749 or TTN expression may also be generated in this fashion.

In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be utilized. Lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region that binds to the target antigen into cells. Where fragments of the antibody are used, the smallest inhibitory fragment that binds to the target antigen often is utilized. For example, peptides having an amino acid sequence corresponding to the Fv region of the antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco et al., Proc. Natl. Acad. Sci. USA 90: 7889-7893 (1993)).

GP6, LAMA4, CHGB, LOC338749 or TTN molecules and compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate GP6, LAMA4, CHGB, LOC338749 or TTN disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of the disorders.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit large therapeutic indices often are utilized. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds often lies within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in a method described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Another example of effective dose determination for an individual is the ability to directly assay levels of “free” and “bound” compound in the serum of the test subject. Such assays may utilize antibody mimics and/or “biosensors” that have been created through molecular imprinting techniques. The compound which is able to modulate GP6, LAMA4, CHGB, LOC338749 or TTN activity is used as a template, or “imprinting molecule”, to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated “negative image” of the compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell et al., Current Opinion in Biotechnology 7: 89-94 (1996) and in Shea, Trends in Polymer Science 2: 166-173 (1994). Such “imprinted” affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example of the use of such matrixes in this way can be seen in Vlatakis, et al., Nature 361: 645-647 (1993). Through the use of isotope-labeling, the “free” concentration of compound which modulates the expression or activity of GP6, LAMAS4, CHGB, LOC338749 or TTN can be readily monitored and used in calculations of IC₅₀. Such “imprinted” affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC₅₀. A rudimentary example of such a “biosensor” is discussed in Kriz et al., Analytical Chemistry 67: 2142-2144 (1995).

Provided herein are methods of modulating GP6, LAMA4, CHGB, LOC338749 or TTN expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method involves contacting a cell with a GP6, LAMA4, CHGB, LOC338749 or TTN or agent that modulates one or more of the activities of GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide activity associated with the cell. An agent that modulates GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-occurring target molecule of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide (e.g., a GP6, LAMA4, CHGB, LOC338749 or TTN substrate or receptor), a GP6, LAMA4, CHGB, LOC338749 or TTN antibody, a GP6, LAMA4, CHGB, LOC338749 or TTN agonist or antagonist, a peptidomimetic of a GP6, LAMA4, CHGB, LOC338749 or TTN agonist or antagonist, or other small molecule.

In one embodiment, the agent stimulates one or more GP6, LAMA4, CHGB, LOC338749 or TTN activities. Examples of such stimulatory agents include active GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide and a nucleic acid molecule encoding GP6, LAMA4, CHGB, LOC338749 or TTN. In another embodiment, the agent inhibits one or more GP6, LAMA4, CHGB, LOC338749 or TTN activities. Examples of such inhibitory agents include antisense GP6, LAMA4, CHGB, LOC338749 or TTN nucleic acid molecules, anti-GP6, LAMA4, CHGB, LOC338749 or TTN antibodies, and GP6, LAMA4, CHGB, LOC338749 or TTN inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, provided are methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) GP6, LAMA4, CHGB, LOC338749 or TTN expression or activity. In a preferred embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that inhibits GP6, LAMA4, CHGB, LOC338749 or TTN expression or activity. In another embodiment, the method involves administering a GP6, LAMA4, CHGB, LOC338749 or TTN polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted GP6, LAMA4, CHGB, LOC338749 or TTN expression or activity.

Stimulation of GP6, LAMA4, CHGB, LOC338749 or TTN activity is desirable in situations in which GP6, LAMA4, CHGB, LOC338749 or TTN is abnormally downregulated and/or in which increased GP6, LAMA4, CHGB, LOC338749 or TTN activity is likely to have a beneficial effect. For example, stimulation of GP6, LAMA4, CHGB, LOC338749 or TTN activity is desirable in situations in which a GP6, LAMA4, CHGB, LOC338749 or TTN is downregulated and/or in which increased GP6, LAMA4, CHGB, LOC338749 or TTN activity is likely to have a beneficial effect. Likewise, inhibition of GP6, LAMA4, CHGB, LOC338749 or TTN activity is desirable in situations in which GP6, LAMA4, CHGB, LOC338749 or TTN is abnormally upregulated and/or in which decreased GP6, LAMA4, CHGB, LOC338749 or TTN activity is likely to have a beneficial effect.

Methods of Treatment

In another aspect, provided are methods for identifying a risk of cancer in an individual as described herein and, if a genetic predisposition is identified, treating that individual to delay or reduce or prevent the development of cancer. Such a procedure can be used to treat breast cancer. Optionally, treating an individual for cancer may include inhibiting cellular proliferation, inhibiting metastasis, inhibiting invasion, or preventing tumor formation or growth as defined herein. Suitable treatments to prevent or reduce or delay breast cancer focus on inhibiting additional cellular proliferation, inhibiting metastasis, inhibiting invasion, and preventing further tumor formation or growth. Treatment usually includes surgery followed by radiation therapy. Surgery may be a lumpectomy or a mastectomy (e.g., total, simple or radical). Even if the doctor removes all of the cancer that can be seen at the time of surgery, the patient may be given radiation therapy, chemotherapy, or hormone therapy after surgery to try to kill any cancer cells that may be left. Radiation therapy is the use of x-rays or other types of radiation to kill cancer cells and shrink tumors. Radiation therapy may use external radiation (using a machine outside the body) or internal radiation. Chemotherapy is the use of drugs to kill cancer cells. Chemotherapy may be taken by mouth, or it may be put into the body by inserting a needle into a vein or muscle. Hormone therapy often focuses on estrogen and progesterone, which are hormones that affect the way some cancers grow. If tests show that the cancer cells have estrogen and progesterone receptors (molecules found in some cancer cells to which estrogen and progesterone will attach), hormone therapy is used to block the way these hormones help the cancer grow. Hormone therapy with tamoxifen is often given to patients with early stages of breast cancer and those with metastatic breast cancer. Other types of treatment being tested in clinical trials include sentinel lymph node biopsy followed by surgery and high-dose chemotherapy with bone marrow transplantation and peripheral blood stem cell transplantation. Any preventative/therapeutic treatment known in the art may be prescribed and/or administered, including, for example, surgery, chemotherapy and/or radiation treatment, and any of the treatments may be used in combination with one another to treat or prevent breast cancer (e.g., surgery followed by radiation therapy).

Also provided are methods of preventing or treating cancer comprising providing an individual in need of such treatment with a GP6, LAMA4, CHGB, LOC338749 or TTN inhibitor that reduces or inhibits the overexpression of mutant GP6, LAMA4, CHGB, LOC338749 or TTN (e.g., a GP6, LAMA4, CHGB, LOC338749 or TTN polynucleotide with an allele that is associated with cancer). Included herein are methods of reducing or blocking the expression of GP6, LAMA4, CHGB, LOC338749 or TTN comprising providing or administering to individuals in need of reducing or blocking the expression of GP6, LAMA4, CHGB, LOC338749 or TTN a pharmaceutical or physiologically acceptable composition comprising a molecule capable of inhibiting expression of GP6, LAMA4, CHGB, LOC338749 or TTN, e.g., a siRNA molecule. Also included herein are methods of reducing or blocking the expression of secondary regulatory genes regulated by GP6, LAMA4, CHGB, LOC338749 or TTN that play a role in oncogenesis which comprises introducing competitive inhibitors that target GP6, LAMA4, CHGB, LOC338749 or TTN's effect on these regulatory genes or that block the binding of positive factors necessary for the expression of these regulatory genes.

The examples set forth below are intended to illustrate but not limit the invention.

EXAMPLES

In the following studies a group of subjects were selected according to specific parameters relating to breast cancer. Nucleic acid samples obtained from individuals in the study group were subjected to genetic analysis, which identified associations between breast cancer and certain polymorphic regions in the GP6, LAMA4, CHGB/C20orf154, LOC338749 and TTN/LOC351327 genes or gene regions (herein referred to as “target genes”, “target nucleotides”, “target polypeptides” or simply “targets”). Methods are described for producing GP6, LAMA4, CHGB/C20orf154, LOC338749, or TTN/LOC351327 polypeptides and polypeptide variants in vitro or in vivo. GP6, LAMA4, CHGB/C20orf154, LOC338749 or TTN/LOC351327 nucleic acids or polypeptides and variants thereof are utilized for screening test molecules for those that interact with GP6, LAMA4, CHGB/C20Orf154, LOC338749 or TTN/LOC351327 molecules. Test molecules identified as interactors with GP6, LAMA4, CHGB/C20orf154, LOC338749 or TTN/LOC351327 molecules and variants are further screened in vivo to determine whether they treat breast cancer.

Example 1 Samples and Pooling Strategies

Sample Selection

Blood samples were collected from individuals diagnosed with breast cancer, which were referred to as case samples. Also, blood samples were collected from individuals not diagnosed with breast cancer as gender and age-matched controls. All of the samples were of German/German descent. A database was created that listed all phenotypic trait information gathered from individuals for each case and control sample. Genomic DNA was extracted from each of the blood samples for genetic analyses.

DNA Extraction from Blood Samples

Six to ten milliliters of whole blood was transferred to a 50 ml tube containing 27 ml of red cell lysis solution (RCL). The tube was inverted until the contents were mixed. Each tube was incubated for 10 minutes at room temperature and inverted once during the incubation. The tubes were then centrifuged for 20 minutes at 3000×g and the supernatant was carefully poured off. 100-200 μl of residual liquid was left in the tube and was pipetted repeatedly to resuspend the pellet in the residual supernatant. White cell lysis solution (WCL) was added to the tube and pipetted repeatedly until completely mixed. While no incubation was normally required, the solution was incubated at 37° C. or room temperature if cell clumps were visible after mixing until the solution was homogeneous. 2 ml of protein precipitation was added to the cell lysate. The mixtures were vortexed vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate, and then centrifuged for 10 minutes at 3000×g. The supernatant containing the DNA was then poured into a clean 15 ml tube, which contained 7 ml of 100% isopropanol. The samples were mixed by inverting the tubes gently until white threads of DNA were visible. Samples were centrifuged for 3 minutes at 2000×g and the DNA was visible as a small white pellet. The supernatant was decanted and 5 ml of 70% ethanol was added to each tube. Each tube was inverted several times to wash the DNA pellet, and then centrifuged for 1 minute at 2000×g. The ethanol was decanted and each tube was drained on clean absorbent paper. The DNA was dried in the tube by inversion for 10 minutes, and then 1000 μl of 1× TE was added. The size of each sample was estimated, and less TE buffer was added during the following DNA hydration step if the sample was smaller. The DNA was allowed to rehydrate overnight at room temperature, and DNA samples were stored at 2-8° C.

DNA was quantified by placing samples on a hematology mixer for at least 1 hour. DNA was serially diluted (typically 1:80, 1:160, 1:320, and 1:640 dilutions) so that it would be within the measurable range of standards. 125 μl of diluted DNA was transferred to a clear U-bottom microtitre plate, and 125 μl of 1× TE buffer was transferred into each well using a multichannel pipette. The DNA and 1× TE were mixed by repeated pipetting at least 15 times, and then the plates were sealed. 50 μl of diluted DNA was added to wells A5-H12 of a black flat bottom microtitre plate. Standards were inverted six times to mix them, and then 50 μl of 1× TE buffer was pipetted into well A1, 1000 ng/ml of standard was pipetted into well A2, 500 ng/ml of standard was pipetted into well A3, and 250 ng/ml of standard was pipetted into well A4. PicoGreen (Molecular Probes, Eugene, Oreg.) was thawed and freshly diluted 1:200 according to the number of plates that were being measured. PicoGreen was vortexed and then 501 was pipetted into all wells of the black plate with the diluted DNA. DNA and PicoGreen were mixed by pipetting repeatedly at least 10 times with the multichannel pipette. The plate was placed into a Fluoroskan Ascent Machine (microplate fluorometer produced by Labsystems) and the samples were allowed to incubate for 3 minutes before the machine was run using filter pairs 485 nm excitation and 538 nm emission wavelengths. Samples having measured DNA concentrations of greater than 450 ng/μl were re-measured for conformation. Samples having measured DNA concentrations of 20 ng/μl or less were re-measured for confirmation.

Pooling Strategies

Samples were placed into one of two groups based on disease status. The two groups were female case groups and female control groups. A select set of samples from each group were utilized to generate pools, and one pool was created for each group. Each individual sample in a pool was represented by an equal amount of genomic DNA. For example, where 25 ng of genomic DNA was utilized in each PCR reaction and there were 200 individuals in each pool, each individual would provide 125 pg of genomic DNA. Inclusion or exclusion of samples for a pool was based upon the following criteria: the sample was derived from an individual characterized as Caucasian; the sample was derived from an individual of German paternal and maternal descent; the database included relevant phenotype information for the individual; case samples were derived from individuals diagnosed with breast cancer; control samples were derived from individuals free of cancer and no family history of breast cancer; and sufficient genomic DNA was extracted from each blood sample for all allelotyping and genotyping reactions performed during the study. Phenotype information included pre- or post-menopausal, familial predisposition, country or origin of mother and father, diagnosis with breast cancer (date of primary diagnosis, age of individual as of primary diagnosis, grade or stage of development, occurrence of metastases, e.g., lymph node metastases, organ metastases), condition of body tissue (skin tissue, breast tissue, ovary tissue, peritoneum tissue and myometrium), method of treatment (surgery, chemotherapy, hormone therapy, radiation therapy). Samples that met these criteria were added to appropriate pools based on gender and disease status.

The selection process yielded the pools set forth in Table 1, which were used in the studies that follow: TABLE 1 Female CASE Female CONTROL Pool size 272 276 (Number) Pool Criteria case control (ex: case/control) Mean Age 59.6 55.4 (ex: years)

Example 2 Association of Polymorphic Variants with Breast Cancer

A whole-genome screen was performed to identify particular SNPs associated with occurrence of breast cancer. As described in Example 1, two sets of samples were utilized, which included samples from female individuals having breast cancer (breast cancer cases) and samples from female individuals not having cancer (female controls). The initial screen of each pool was performed in an allelotyping study, in which certain samples in each group were pooled. By pooling DNA from each group, an allele frequency for each SNP in each group was calculated. These allele frequencies were then compared to one another. Particular SNPs were considered as being associated with breast cancer when allele frequency differences calculated between case and control pools were statistically significant. SNP disease association results obtained from the allelotyping study were then validated by genotyping each associated SNP across all samples from each pool. The results of the genotyping were then analyzed, allele frequencies for each group were calculated from the individual genotyping results, and a p-value was calculated to determine whether the case and control groups had statistically significantly differences in allele frequencies for a particular SNP. When the genotyping results agreed with the original allelotyping results, the SNP disease association was considered validated at the genetic level.

SNP Panel Used for Genetic Analyses

A whole-genome SNP screen began with an initial screen of approximately 25,000 SNPs over each set of disease and control samples using a pooling approach. The pools studied in the screen are described in Example 1. The SNPs analyzed in this study were part of a set of 25,488 SNPs confirmed as being statistically polymorphic as each is characterized as having a minor allele frequency of greater than 10%. The SNPs in the set reside in genes or in close proximity to genes, and many reside in gene exons. Specifically, SNPs in the set are located in exons, introns, and within 5,000 base-pairs upstream of a transcription start site of a gene. In addition, SNPs were selected according to the following criteria: they are located in ESTs; they are located in Locuslink or Ensemble genes; and they are located in Genomatix promoter predictions. SNPs in the set also were selected on the basis of even spacing across the genome, as depicted in Table 2.

A case-control study design using a whole genome association strategy involving approximately 28,000 single nucleotide polymorphisms (SNPs) was employed. Approximately 25,000 SNPs were evenly spaced in gene-based regions of the human genome with a median inter-marker distance of about 40,000 base pairs. Additionally, approximately 3,000 SNPs causing amino acid substitutions in genes described in the literature as candidates for various diseases were used. The case-control study samples were of female German origin (German paternal and maternal descent) 548 individuals were equally distributed in two groups (female controls and female cases). The whole genome association approach was first conducted on 2 DNA pools representing the 2 groups. Significant markers were confirmed by individual genotyping. TABLE 2 General Statistics Spacing Statistics Total # of SNPs 25,488 Median  37,058 bp # of Exonic SNPs >4,335 (17%) Minimum*  1,000 bp # SNPs with refSNP ID 20,776 (81%) Maximum* 3,000,000 bp  Gene Coverage >10,000 Mean 122,412 bp Chromosome Coverage All Std 373,325 bp Deviation *Excludes outliers Allelotyping and Genotyping Results

The genetic studies summarized above and described in more detail below identified allelic variants associated with breast cancer. The allelic variants identified from the SNP panel described in Table 2 are summarized below in Table 3. TABLE 3 SNP Chromosome Position in Contig Contig Sequence Sequence Allelic Reference Position FIGS. 1-4 Identification Position Identification Position Variability rs1671152 60202366 45666 NT_011109 27794535 NM_016363 exonic T/G (T323K) rs1050348 112494002 47502 NT_025741 16663301 NM_002290 exonic C/T (H491Y) rs454422 5891693 49293 NT_011387 5883693 NM_032485 intragenic A/C rs763471 10491273 49273 NT_009237 1853223 G/T rs2046778 179636570 49170 NT_005403 29831884 X90569 upstream A/G

Table 3 includes information pertaining to the incident polymorphic variant associated with breast cancer identified herein. Public information pertaining to the polymorphism and the genomic sequence that includes the polymorphism are indicated. The genomic sequences identified in Table 3 ay be accessed at the http address www.ncbi.nih.gov/entrez/query.fcgi, for example, by using the publicly available SNP reference number. The chromosome position refers to the position of the SNP within NCBI's Genome Build 33, which may be accessed at the following http address: www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=hum_chr.inf&query=. The “Contig Position” provided in Table 3 corresponds to a nucleotide position set forth in the contig sequence, and designates the polymorphic site corresponding to the SNP reference number. The sequence containing the polymorphisms also may be referenced by the “Sequence Identification” set forth in Table 3. The “Sequence Identification” corresponds to cDNA sequence that encodes associated target polypeptides of the invention. The position of the SNP within the cDNA sequence is provided in the “Sequence Position” column of Table 3. Also, the allelic variation at the polymorphic site and the allelic variant identified as associated with breast cancer is specified in Table 3. All nucleotide sequences referenced and accessed by the parameters set forth in Table 3 are incorporated herein by reference.

Assay for Verifying, Allelotyping and Genotyping SNPs

A MassARRAY™ system (Sequenom, Inc.) was utilized to perform SNP genotyping in a high-throughput fashion. This genotyping platform was complemented by a homogeneous, single-tube assay method (hME™ or homogeneous MassEXTEND™ (Sequenom, Inc.)) in which two genotyping primers anneal to and amplify a genomic target surrounding a polymorphic site of interest. A third primer (the MassEXTEND™ primer), which is complementary to the amplified target up to but not including the polymorphism, was then enzymatically extended one or a few bases through the polymorphic site and then terminated.

For each polymorphism, SpectroDESIGNER™ software (Sequenom, Inc.) was used to generate a set of PCR primers and a MassEXTEND™ primer was used to genotype the polymorphism. Table 4 shows PCR primers and Table 5 shows extension primers used for analyzing polymorphisms. The initial PCR amplification reaction was performed in a 5 μl total volume containing 1× PCR buffer with 1.5 mM MgCl₂ (Qiagen), 200 μM each of dATP, dGTP, dCTP, dTTP (Gibco-BRL), 2.5 ng of genomic DNA, 0.1 units of HotStar DNA polymerase (Qiagen), and 200 nM each of forward and reverse PCR primers specific for the polymorphic region of interest. TABLE 4 PCR Primers Reference Forward Reverse SNP ID PCR primer PCR primer rs1671152 ACGTTGGATGAGGGCTGTGCAGAGGCCGCTT ACGTTGGATGTGAACATCCTGTCGGCCTCC rs1050348 CAGCTGGATGACTACAATGC GTTCATGTCTTCGGCATCC rs454422 CAGCTTTTGAGGCACTTTCC AGCACCTTGCATACCCATAG rs763471 TAACTCCTGTGTGGCTTTCT GTGAAGAGCTCTGAAATGCC rs2046778 CATGAAGCCTTATGCTTGAG GTTCCCTTCCCCCATAAAAC

Samples were incubated at 95° C. for 15 minutes, followed by 45 cycles of 95° C. for 20 seconds, 56° C. for 30 seconds, and 72° C. for 1 minute, finishing with a 3 minute final extension at 72° C. Following amplification, shrimp alkaline phosphatase (SAP) (0.3 units in a 2 μl volume) (Amersham Pharmacia) was added to each reaction (total reaction volume was 7 μl) to remove any residual dNTPs that were not consumed in the PCR step. Samples were incubated for 20 minutes at 37° C., followed by 5 minutes at 85° C. to denature the SAP.

Once the SAP reaction was complete, a primer extension reaction was initiated by adding a polymorphism-specific MassEXTEND™ primer cocktail to each sample. Each MassEXTEND™ cocktail included a specific combination of dideoxynucleotides (ddNTPs) and deoxynucleotides (dNTPs) used to distinguish polymorphic alleles from one another. In Table 5, ddNTPs are shown and the fourth nucleotide not shown is the dNTP. TABLE 5 Extend Primers Reference Extend Term SNP ID Probe Mix rs1671152 CTCCATCCTGACCCCGT ACT rs1050348 CACTTGACCAGGCCCTTAAC ACG rs454422 GATCCTTCTCACTTACTGTTC ACT rs763471 CTCCAAGCAGTAAAGATGTTC CGT rs2046778 CTGTCATGATTGACAGGTCC ACT

The MassEXTEND™ reaction was performed in a total volume of 9 μl, with the addition of 1× ThermoSequenase buffer, 0.576 units of ThermoSequenase (Amersham Pharmacia), 600 nM MassEXTEND™ primer, 2 mM of ddATP and/or ddCTP and/or ddGTP and/or ddTTP, and 2 mM of dATP or dCTP or dGTP or dTTP. The deoxy nucleotide (dNTP) used in the assay normally was complementary to the nucleotide at the polymorphic site in the amplicon. Samples were incubated at 94° C. for 2 minutes, followed by 55 cycles of 5 seconds at 94° C., 5 seconds at 52° C., and 5 seconds at 72° C.

Following incubation, samples were desalted by adding 16 μl of water (total reaction volume was 25 μl), 3 mg of SpectroCLEAN™ sample cleaning beads (Sequenom, Inc.) and allowed to incubate for 3 minutes with rotation. Samples were then robotically dispensed using a piezoelectric dispensing device (SpectroJET™ (Sequenom, Inc.)) onto either 96-spot or 384-spot silicon chips containing a matrix that crystallized each sample (SpectroCHIP® (Sequenom, Inc.)). Subsequently, MALDI-TOF mass spectrometry (Biflex and Autoflex MALDI-TOF mass spectrometers (Bruker Daltonics) can be used) and SpectroTYPER RT™ software (Sequenom, Inc.) were used to analyze and interpret the SNP genotype for each sample.

Genetic Analysis

Variations identified in the target genes are provided in their respective genomic sequences (see FIGS. 1-5) Minor allelic frequencies for these polymorphisms was verified as being 10% or greater by determining the allelic frequencies using the extension assay described above in a group of samples isolated from 92 individuals originating from the state of Utah in the United States, Venezuela and France (Coriell cell repositories).

Genotyping results are shown for female pools in Table 6A and 6B. Table 6A shows the orginal genotyping results and Table 6B shows the genotyped results re-analyzed to remove duplicate individuals from the cases and controls (i.e., individuals who were erroneously included more than once as either cases or controls). Therefore, Table 6B represents a more accurate measure of the allele frequencies for this particular SNP. In the subsequent tables, “AF” refers to allelic frequency; and “F case” and “F control” refer to female case and female control groups, respectively. TABLE 6A Breast Reference AF AF Cancer SNP ID F case F control p-value Assoc. Allele rs1671152 T = 0.139 T = 0.192 0.0196 G G = 0.861 G = 0.808 rs1050348 C = 0.625 C = 0.540 0.0050 C T = 0.375 T = 0.460 rs454422 C = 0.831 C = 0.761 0.0049 C A = 0.169 A = 0.239 rs763471 T = 0.523 T = 0.593 0.0079 G G = 0.477 G = 0.407 rs2046778 A = 0.836 A = 0.736 0.0019 A G = 0.164 G = 0.264

TABLE 6B Breast Cancer Reference AF AF Assoc. SNP ID F case F control p-value Odds Ratio Allele rs1671152 T = 0.143 T = 0.190 0.00109 0.65 G G = 0.857 G = 0.810 (T323K) rs1050348 C = 0.629 C = 0.543 0.0124 0.72 C T = 0.371 T = 0.457 (H491Y) rs454422 C = 0.834 C = 0.762 0.00452 1.57 C A = 0.166 A = 0.238 rs763471 T = 0.520 T = 0.596 0.0166 0.74 G G = 0.480 G = 0.404 rs2046778 A = 0.811 A = 0.724 0.00114 0.61 A G = 0.189 G = 0.276

The single marker alleles set forth in Table 3 were considered validated, since the genotyping data for the females, males or both pools were significantly associated with breast cancer, and because the genotyping results agreed with the original allelotyping results. Particularly significant associations with breast cancer are indicated by a calculated p-value of less than 0.05 for genotype results, which are set forth in bold text. Tables 6A and 6B show the disease associated allele in column 6. In the case of rs1671152, this SNP is an exonic SNP that codes for a T323K amino acid change in the GP6 gene. The guanine allele codes for threonine (T); therefore, a threonine is associated with an increased risk of breast cancer. In the case of rs454422, this SNP is an exonic SNP that codes for a H491Y amino acid change in the LAMA4 gene. The cytosine allele codes for histidine (H); therefore, a histidine is associated with an increased risk of breast cancer.

Odds ratio results are shown in Tables 6B. An odds ratio is an unbiased estimate of relative risk which can be obtained from most case-control studies. Relative risk (RR) is an estimate of the likelihood of disease in the exposed group (susceptibility allele or genotype carriers) compared to the unexposed group (not carriers). It can be calculated by the following equation: RR=IA/Ia

IA is the incidence of disease in the A carriers and Ia is the incidence of disease in the non-carriers.

RR>1 indicates the A allele increases disease susceptibility.

RR<1 indicates the a allele increases disease susceptibility.

For example, RR=1.5 indicates that carriers of the A allele have 1.5 times the risk of disease than non-carriers, i.e., 50% more likely to get the disease.

Case-control studies do not allow the direct estimation of IA and Ia, therefore relative risk cannot be directly estimated. However, the odds ratio (OR) can be calculated using the following equation: OR=(nDAnda)/(ndAnDa)=pDA(1−pdA)/pdA(1−pDA), or OR=((case f)/(1−case f))/((control f)/(1−control f)), where f=susceptibility allele frequency.

An odds ratio can be interpreted in the same way a relative risk is interpreted and can be directly estimated using the data from case-control studies, i.e., case and control allele frequencies. The higher the odds ratio value, the larger the effect that particular allele has on the development of breast cancer. Possessing an allele associated with a relatively high odds ratio translates to having a higher risk of developing or having breast cancer.

Example 3 GP6 Region Proximal SNPs

It has been discovered that a polymorphic variation (rs1671152) in a region that encodes the glycoprotein VI (platelet)(GP6) gene is associated with the occurrence of breast cancer (see Examples 1 and 2). Subsequently, SNPs proximal to the incident SNP (rs1671152) were identified and allelotyped in breast cancer sample sets and control sample sets as described in Examples 1 and 2. Approximately 124 allelic variants located within or near the GP6 gene were identified and 114 SNPs were allelotyped. The polymorphic variants are set forth in Table 7. The chromosome position provided in column four of Table 7 is based on Genome “Build 33” of NCBI's GenBank. TABLE 7 dbSNP Position in Chromosome Allele rs# Chromosome Position Variants 269911 19 185 60156885 A/T 703464 19 237 60156937 G/A 703465 19 641 60157341 C/G 269912 19 719 60157419 A/G 269913 19 990 60157690 T/C 269915 19 2908 60159608 C/T 269916 19 3140 60159840 G/A 172006 19 3880 60160580 A/G 703467 19 4494 60161194 C/T 703468 19 5107 60161807 G/A 2217659 19 5220 60161920 G/A 775894 19 6031 60162731 A/G 775900 19 8670 60165370 A/G 1654491 19 13794 60170494 T/C 775903 19 16356 60173056 A/G 1036231 19 17164 60173864 T/C 1036232 19 17264 60173964 G/A 1671223 19 20537 60177237 T/G 1671224 19 20637 60177337 A/G 1654495 19 20900 60177600 C/A 1654496 19 21155 60177855 C/A 1654497 19 21795 60178495 T/C 1671225 19 21931 60178631 T/G 1671226 19 22167 60178867 C/G 1671227 19 22656 60179356 T/C 1654498 19 23108 60179808 T/C 1671228 19 23404 60180104 T/C 1654499 19 24287 60180987 T/C 1869616 19 24480 60181180 A/C 1654503 19 24592 60181292 C/T 1654504 19 24878 60181578 T/C 1671133 19 26370 60183070 A/C 1654505 19 27056 60183756 G/A 3786863 19 27874 60184574 A/G 1560714 19 31248 60187948 G/A 1654406 19 31458 60188158 G/T 1043673 19 31553 60188253 C/A 1043678 19 31637 60188337 G/T 1043680 19 31668 60188368 C/G 1043684 19 31752 60188452 A/G 1671140 19 37643 60194343 G/A 1654409 19 43941 60200641 A/C 1654410 19 44134 60200834 T/C 1654411 19 44329 60201029 A/C 1671148 19 44343 60201043 A/C 1671149 19 44362 60201062 G/A 1671150 19 44818 60201518 G/A 1654412 19 44917 60201617 C/T 1671151 19 45215 60201915 G/A 1671152 19 45666 60202366 T/G 1654413 19 45680 60202380 T/A 2304167 19 46402 60203102 C/T 1671153 19 46510 60203210 G/T 2019599 19 46554 60203254 C/T 1654485 19 46823 60203523 A/C 1671188 19 47714 60204414 T/G 1671191 19 48963 60205663 T/C 1654415 19 49157 60205857 C/T 1671192 19 49254 60205954 G/A 2304168 19 49257 60205957 A/G 1654416 19 49356 60206056 T/C 1654419 19 55202 60211902 A/G 1654420 19 55527 60212227 T/A 1613662 19 55916 60212616 G/A 2886415 19 56402 60213102 T/G 2365593 19 56413 60213113 C/T 2886414 19 56685 60213385 G/A 1654421 19 56783 60213483 A/G 1654424 19 58044 60214744 A/G 1654425 19 58301 60215001 T/C 892089 19 58382 60215082 A/G 892090 19 58393 60215093 G/T 1671196 19 58869 60215569 C/T 1671198 19 59155 60215855 T/C 1671199 19 59189 60215889 G/A 1625609 19 62546 60219246 C/T 1625689 19 62568 60219268 G/A 1654438 19 70983 60227683 A/G 2569513 19 71465 60228165 G/A 2569514 19 71538 60228238 G/A 1671214 19 72144 60228844 G/A 1671215 19 72340 60229040 C/A 1054796 19 72527 60229227 C/G 1654439 19 72968 60229668 T/G 1671216 19 73397 60230097 A/G 1671217 19 73553 60230253 G/A 1671218 19 73720 60230420 C/T 1654441 19 74190 60230890 C/T 1654442 19 74687 60231387 T/G 1671219 19 74699 60231399 G/A 10666 19 75580 60232280 C/T 1626971 19 76345 60233045 T/C 1671221 19 76506 60233206 G/A 754235 19 77554 60234254 G/A 775821 19 77889 60234589 C/T 3745912 19 77919 60234619 A/G 775822 19 78866 60235566 A/G 1059211 19 79061 60235761 C/T 2124090 19 83777 60240477 A/C 1671171 19 84360 60241060 T/G 1671170 19 84631 60241331 T/A 1654444 19 85775 60242475 G/T 2365721 19 87153 60243853 G/A 1654446 19 89650 60246350 A/G 1654447 19 89895 60246595 A/G 1671176 19 90103 60246803 C/A 1654448 19 90234 60246934 G/A 1671178 19 90309 60247009 G/A 1654449 19 90376 60247076 G/A 1671182 19 90925 60247625 C/T 1654451 19 91561 60248261 A/T 1654452 19 91605 60248305 G/A 1671169 19 92954 60249654 T/C 1654459 19 94228 60250928 A/G 269909 19 Not Mapped G/C 269910 19 Not Mapped T/G 776251 19 Not Mapped G/A 892088 19 Not Mapped A/G 892091 19 Not Mapped C/G 1043680 19 Not Mapped C/G 1064675 19 Not Mapped A/G 1671187 19 Not Mapped T/A 2116883 19 Not Mapped T/C 2163833 19 Not Mapped G/A Assay for Verifying and Allelotyping SNPs

The methods used to verify and allelotype the proximal SNPs of Table 7 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 8 and Table 9, respectively. TABLE 8 dbSNP Forward Reverse rs# PCR primer PCR primer 10666 ACGTTGGATGAGATGGCCCCTCTCCCCT ACGTTGGATGAGTGGACTGGCCTGCAGGT 172006 ACGTTGGATGGGTTGAGGAGTATTTCCATTG ACGTTGGATGTGGGTGACAGCAAGACTCCA 269909 AACGTTGGATGAGTTTCTTGTCTCCTGGGTG ACGTTGGATGCACAACAATGAAAGGACAAGC 269910 ACGTTGGATGGGCTGCTCAGGTTCTAAAAG ACGTTGGATGCCCCAGTTCCTTATCTGATC 269911 ACGTTGGATGGGTGACAAAGTGAGACTCCG ACGTTGGATGTGACTAGCTGGGATTATGGG 269912 ACGTTGGATGAGGAGAGCCTGCAGGTTGAA ACGTTGGATGTCCCTTGATTGTCATCACAG 269913 ACGTTGGATGAGATGCACCGAATGGATCTG ACGTTGGATGTCCTAGGACACAGTGTGGAC 269915 ACGTTGGATGAAGCTGAGATTGTGCTGCTG ACGTTGGATGACTACTCTACTTTCTACCCC 269916 ACGTTGGATGAACTCCTGACCACGTGATCC ACGTTGGATGAAAGTGTAGCTGGGCATGGC 703464 ACGTTGGATGAAAAGTAGCTGAGCGTGGTG ACGTTGGATGCCCAGGTTCAAACAGTTCTC 703465 ACGTTGGATGGCTGTGATGACAATCkAGGG ACGTTGGATGAGCAGGAACTGGCATTTGAG 703467 ACGTTGGATGAAACTTACGAAGGTCTGGGC ACGTTGGATGGGGTTTCAGGAACATTCACC 703468 ACGTTGGATGGAAAGGAACAAGTGATCCAG ACGTTGGATGCTTCTGAAAAAGGAGAAGGG 754235 ACGTTGGATGGTGGAACAAACAGTTGGAGC ACGTTGGATGGAGTGGAGATCATGCCATTG 775821 ACGTTGGATGTTTTCTCATCCGCCAGCAGG ACGTTGGATGACAGAGCACAGGTCCCTTTC 775822 ACGTTGGATGAAAAGTAGAGCATGTGGACC ACGTTGGATGGGGATGAACAGACAATCCTC 775894 ACGTTGGATGAGGCAGGAGGACTGATTGAG ACGTTGGATGTTGTAGAGACAGGGTCTTGC 775900 ACGTTGGATGTCTGCAACGTTCGTTCTTCC ACGTTGGATGTTCTTAATGTGCCCACTGTC 775903 ACGTTGGATGTGTGCCCGGCCCTTTTTTTC ACGTTGGATGGGACTCCAGTCTGAGTGACA 776251 ACGTTGGATGAATCCTAGCTACTCAGGAGG ACGTTGGATGATGGTATGATCTAGGCTCCC 892088 ACGTTGGATGATTGGTTACACTGGGACTGC ACGTTGGATGAAGCGGTGATTCTCAGCCTC 892089 ACGTTGGATGCCCTACAAGAATCCCGAGAG ACGTTGGATGAAGCTGTAGCATCGGTAGGTT 892090 ACGTTGGATGAAGCTGTAGCATCGGTAGGTT ACGTTGGATGCCCTACAAGAATCCCGAGAG 892091 ACGTTGGATGAAGATGGAGGACCACAGGTG ACGTTGGATGACCGGAATTACTGAGAGGTC 1036231 ACGTTGGATGAACAGTACTAAGGGCAGATG ACGTTGGATGGTCCAGGGTGTTTACTGTTC 1036232 ACGTTGGATGCCAAAGAAACTCCCAGAATC ACGTTGGATGGATCGTGCCATTGCACTTTG 1043673 ACGTTGGATGTGAAGTCATGAGAAGPAGGC ACGTTGGATGGCTGCTGGAAGAAATAGAAG 1043678 ACGTTGGATGTTCATGATCTGAATCCCCCC ACGTTGGATGGAACTCATTATCCCTGAGGG 1043680 ACGTTGGATGTCTAGCCCAGCAATGAACTC ACGTTGGATGTTCCAGGTGTTGGTGAACTG 1043684 ACGTTGGATGCTCAATATACCCGTGATACAG ACGTTGGATGTTTAGCCATGATTCTGCCTC 1054796 ACGTTGGATGAGTGTCACCCTGATTTCCAG ACGTTGGATGCACCTTGGGAAATACGTTGC 1059211 ACGTTGGATGCTCAGCTCCTTGGTGAAGAG ACGTTGGATGGGCAGACGAGGAAGTATAAC 1064675 ACGTTGGATGGAGTTCCCTCAGTTTTTATTG ACGTTGGATGCCTACTACATTCCTTTTTTGC 1560714 ACGTTGGATGATCTGCTGACCTCGTGATCC ACGTTGGATGAAAAGACAGTCTCAGGTGGG 1613662 ACGTTGGATGATGGACCCTGCAGAACCTAC ACGTTGGATGTCTGATTTCCCAGGAACCTC 1625609 ACGTTGGATGTCAAGCGATTCTCCTGCCTC ACGTTGGATGAAAAAATGAGCTGGGCGTGG 1625689 ACGTTGGATGGTAATCCCAGCTACTTGGAG ACGTTGGATGATCTTGGCTCACTGCAGCCT 1626971 ACGTTGGATGTATTAAATGCACCTGGCACC ACGTTGGATGCAAAGTGCTGGGATTACAGG 1654406 ACGTTGGATGCTTCTATTTCTTCCAGCAGC ACGTTGGATGTTTCTTCCCCCATTGTACCC 1654409 ACGTTGGATGGTGAAACCTTGTCTCATATAC ACGTTGGATGTGAGAGTAGGCATGTGGTAC 1654410 ACGTTGGATGACTGTGCCTAGGCTATACTG ACGTTGGATGGGAAAATCATACTGAGATGC 1654411 ACGTTGGATGTGGAACTTCTTGGTGCCATC ACGTTGGATGCCTGTAATCCCGGCACTTTG 1654412 ACGTTGGATGATTAGCCAGGTGTGGTGGTG ACGTTGGATGTCAAGCCATTCTCCCACCTC 1654413 ACGTTGGATGAGGGCTGTGCAGAGGCCGCTT ACGTTGGATGTCCATCCTGACCCCCGTTTG 1654415 ACGTTGGATGCCAAGAAAGTCCTTGGTGTG ACGTTGGATGCTTTGAAATGGCCCCATCAC 1654416 ACGTTGGATGTCTGCTGAGCATGAAATGCC ACGTTGGATGCTGAACTGACCGTCTCATTC 1654419 ACGTTGGATGTATCATACGCTAGGCTGGAG ACGTTGGATGATGTTTCTCCTGCCTTGGTG 1654420 ACGTTGGATGCCAACCAACCAACAAACCTG ACGTTTGGATGTGGAAGTTTGAGAACCGCTG 1654421 ACGTTGGATGAGGACACAGGAATCCAGAAG ACGTTGGATGGCACATTCTGGGCTATTAAC 1654424 ACGTTGGATGTAGGTGGGAAGGAAGTGGGA ACGTTGGATGCCACTTCTTTCCCACCTATG 1654425 ACGTTGGATGTACCTGTGACCACAAGCTCC ACGTTGGATGTGCTACAGCTTCTCCAGCAG 1654438 ACGTTGGATGAATCAACTAGGCATGGTGGC ACGTTGGATGCCAGGTTCAAGCGATTCTCC 1654439 ACGTTGGATGCCCCATATACATGTGCGATG ACGTTGGATGAATGGGGTGTTTTCTGGAGCA 1654441 ACGTTGGATGAGTAGCTGGGATTACAGGCG ACGTTGGATGGGAGTTCAAGATAAGCCTGG 1654442 ACGTTGGATGAGGAGAATGGTGTGAAGCTG ACGTTGGATGAATCTTGCTCTGTCACCCAG 1654444 ACGTTGGATGGGATGGTCCCAGTTTTACAT ACGTTGGATGCCAGGAGAATCACTTTTATGG 1654446 ACGTTGGATGAAAAGGAAGGGCATTCTGGC ACGTTGGATGTTTGGCCTCCCAAAGTACTG 1654447 ACGTTGGATGATCCCTGGGAAGACGGTCAT ACGTTGGATGTTACCTCTCCTGGCCAGTTC 1654448 ACGTTGGATGTGCTCACTGCATGAGATTCC ACGTTGGATGAACTTTGGCCTCCCAAAGTG 1654449 ACGTTGGATGAGTCCAGCCTGGCAAACATG ACGTTGGATGCAGTCTAATCTCTCTTTTCCC 1654451 ACGTTGGATGTTTAAPATGCCCGCTGCACG ACGTTGGATGAGGAGGATGCACTTATGTGG 1654452 ACGTTTGGATGCTGTACGCATTACCACAGAC ACGTTGGATGGTTTTGGACTCTTGACCTGC 1654459 ACGTTGGATGCAGGAGCTTGGGTACCCAC ACGTTGGATGCCCTCATCTGGAAATGTGTG 1654485 ACGTTGGATGTTGTACCACTGCACTCTAGC ACGTTGGATGCCTGACTCTACAGTTCTTGC 1654491 ACGTTGGATGCAGACGTCCGTGCTTTCACC ACGTTTGGATGTCCAGGAACAGACGGAGGTC 1654495 ACGTTGGATGATGACCATTGCTCGTCTGTG ACGTTGGATGGCTTTCTGCAGAGGTTGTCG 1654496 ACGTTGGATGAATCACAAATGGCAACACGG ACGTTGGATGTTTGGATGCTGGCACTTGTG 1654497 ACGTTGGATGACCCCATGCTGTGTTTTCTC ACGTTGGATGCAGAAGACTACCTGATTTGC 1654498 ACGTTGGATGCTTCCCACACCCACTATATC ACGTTGGATGGTTAGTGAGTCGGTGACATC 1654499 ACGTTGGATGCACTACCTCTCTAGCAACTG ACGTTGGATGACCTCAGATGATCTGCCCAC 1654503 ACGTTGGATGTCCTTGGCTTGTGGCCCTTC ACGTTGGATGAGCCAGGGCAACGTTTGAAG 1654504 ACGTTGGATGCCACCCCATGATTCCATTTC ACGTTGGATGTGCTGTGATGCACCTTTGAC 1654505 ACGTTGGATGCCCTGTCTCTCTAAAACCAC ACGTTTGGATGATTCAAGCAGTTCTCGTGCC 1671133 ACGTTGGATGGTGGTCTCAACTTGGCTATC ACGTTGGATGCCAGATAGGATTCCAGGTTC 1671140 ACGTTGGATGAGTCTGACAAGAGAGTCAGC ACGTTGGATGTCCTTTACCTACCCACATCC 1671148 ACGTTGGATGGCCATCCTTCTGTCTTTTCC ACGTTGGATGAGTGGCTCATGCCTGTAATC 1671149 ACGTTGGATGCTTTTCCCAAGTGACTCACC ACGTTGGATGAAAAGAATGGCTGGCCACAG 1671150 ACGTTGGATGGTGCTATGATCAAATCAGGG ACGTTGGATGACACCACTGCACTCTAGCTC 1671151 ACGTTGGATGGGAAAACCAGACAAGAGCAC ACGTTGGATGTGACTCTGTTCCATCCTCTG 1671152 ACGTTGGATGAGGGCTGTGCAGAGGCCGCTT ACGTTGGATGTGAACATCCTGTCGGCCTCC 1671153 ACGTTGGATGCCTACTCCGAACACACACAC ACGTTGGATGATTATAGGCATGAGGCACCG 1671169 ACGTTGGATGTCCTGTTGCTGGACACTATC ACGTTGGATGTCACACCTTCCGAGGATTTAG 1671170 ACGTTGGATGAGGTGACAGTGCTGTACCTG ACGTTGGATGACAAAGAACAGTGAGAGGGC 1671171 ACGTTGGATGAAGCAAGATACCGTCTCAGA ACGTTGGATGCCGGGAAATGGAATAATTCC 1671176 ACGTTGGATGTGGAGCCACTTATGGAGAAC ACGTTGGATGACCCCAACTGAAACACAGAC 1671178 ACGTTGGATGTAATCCCAGCACTTTGGGAG ACGTTGGATGCATGTTTGCCAGGCTGGACT 1671182 ACGTTGGATGATAGGGCGGCTTTTCTCCTG ACGTTGGATGCCTGGGAACTGAATGTCTCG 1671187 ACGTTGGATGAGTGCTCAGCAACGATTACG ACGTTGGATGGAGGGCTGCAGGTTGAGAAA 1671188 ACGFTTGGATGGGAACCGCAGATGGACAATG ACGTTGGATGAGATCACAGAGTGAGGAGAG 1671191 ACGTTGGATGTCGGACGCACACAGACTGTAG ACGTTGGATGGGAAAGCGTATCTGCAGAGG 1671192 ACGTTGGATGTGGTAAGAGACGGACAGTTC ACGTTGGATGTCAGCAGAAAGGAGTGTGAG 1671196 ACGTTGGATGTTGCTAGGCAACAGGCACTC ACGTTGGATGTCTGTATCTGAGCCTCACTG 1671198 ACGTTGGATGATGAAACTAAGGCACATGGC ACGTTGGATGCTTATAATCTACCCTCTTAGC 1671199 ACGTTGGATGGCTGAAATTTGCTAAGAGGG ACGTTGGATGGACAGTTACTACTAGCAAGC 1671214 ACGTTGGATGAGGCGGAGAATGATCCGGTG ACGTTGGATGACGCCATCATTCGTGCATCC 1671215 ACGTTGGATGTTCTCCAAAGCACCCAAGTG ACGTTGGATGATGCTGGGCTTGCTTTTTCC 1671216 ACGTTTGGATGTGCTTGGGAGCAAGTTACAG ACGTTGGATGTTCCCCCTCCTGGTATTTAC 1671217 ACGTTGGATGTTGTCTCCATTCCTCCCTGG ACGTTGGATGTCTTGTCTTGCCCTCTCGCT 1671218 ACGTTGGATGTGAGTCTGGTAGGCAACTTC ACGTTGGATGTAGAAGCCAGTCGCTACATC 1671219 ACGTTGGATGTGATCTCGGCTCACTGCAAG ACGTTGGATGAAATTAGCTGGGCATGGTGG 1671221 ACGTTGGATGTGGTGAAACCCCATTTCTAC ACGTTGGATGGGTTCAAGGGATTCTCCTGC 1671223 ACGTTGGATGTCAAGTGATTCTCCTGCCTC ACGTTGGATGCCCACCTCTACTGAAAATAC 1671224 ACGTTGGATGTGAGTCTCACTCTTGTTGCC ACGTTGGATGCAGGAGAATCACTTTGAACCC 1671225 ACGTTGGATGTATAGGCGTGAGCCACTATG ACGTTGGATGCTATTGGAAGCTACATGCTC 1671226 ACGTTGGATGTATTGGCCAGACTGGACTTC ACGTTGGATGAGTTACTCAGGAGGCTAAGG 1671227 ACGTTGGATGGGTTTCTGTTCAGAGATTCG ACGTTGGATGTGCAGTGAGCCTAGATCATG 1671228 ACGTTGGATGTCAGCCTCCCAGGGATTAAG ACGTTGGATGACATGGTGAAAACTCGTCTC 1869616 ACGTTGGATGTAATCCCAGCTACTCGGAAG ACGTTGGATGACGGTGGCTCACTTTCAACCT 2019599 ACGTTGGATGGTGCTGGGATTATAGGCATG ACGTTGGATGTACTCCGAACACACACACAC 2116883 ACGTTGGATGATTACAGGCATGAGCCACTG ACGTTGGATGCACGCGCAGTTCAATTTCTC 2124090 ACGTTGGATGTCTGACAAAGCTGGAAGCTG ACGTTGGATGCTGATAAACAAGGCTGTGGG 2163833 ACGTTGGATGGATATTGGTGAGTATGCAGAG ACGTTGGATGAACTGTTTTCCACAGCAGGG 2217659 ACGTTGGATGTTCCCCCCTTCTCCTTTTTC ACGTTGGATGATGAGGTAACTTACCTTATG 2304167 ACGTTGGATGGTTTGGTTCCCAGAGACTTC ACGTTGGATGAGGATGACTTACTCACCAGC 2304168 ACGTTTGGATGTCAGCAGAAAGGAGTGTGAG ACGTTGGATGTGGTAAGAGACGGACAGTTC 2365593 ACGTTGGATGTGACGCAGTAAGACTCCATC ACGTTGGATGCAAAGTGCTGGGATTACAGG 2365721 ACGTTGGATGTTGTACAGCCTGCAAGCAAC ACGTTGGATGAGATCGCGCCATTGCACTCA 2569513 ACGTTGGATGGTTGGCGTTTTTGTTTGCAC ACGTTGGATGTCTCATAGTATTCTGCAGGG 2569514 ACGTTGGATGTCCCTGCAGAATACTATGAG ACGTTGGATGAGAGTGTTGGGATTACAGGC 2886414 ACGTTGGATGGGTGTGCTTTACAAATGCTG ACGTTGGATGAACTGAGATCACTCCACTGC 2886415 ACGTTGGATGTGACGCAGTAAGACTCCATC ACGTTGGATGCAAAGTGCTGGGATTACAGG 3745912 ACGTTGGATGACGTCTTCTGAGGCACAGAG ACGTTGGATGGCTGTTAGAGGCTGGCAGG 3786863 ACGTTGGATGTGACCAACAGAAGTCTCAGG ACGTTGGATGTTGACCTCAGGTGATCCATC

TABLE 9 dbSNP Extend Term rs# Primer Mix 10666 TGCAGGTGAGCACTGCCC ACG 172006 GCAAGACTCCATCTCAA ACT 269909 AAGCATAGATCAGATAAGGAA ACT 269910 ATCTATGCTTGTCCTTTCAT ACT 269911 GCTCAGCTACTTTTTGTAT CGT 269912 CAAGATGGTGTCTTCGGC ACT 269913 ACAGTGTGGACCGATTTCC ACT 269915 AGACAAGTCTCACTCTG ACG 269916 GGCGGCTCACACCTGTAAT ACG 703464 CTCCTGCCTCAGCCTCC ACG 703465 TGGCATTTGAGACAGGA ACT 703467 CATTCACCATGTCTGTGTGAG ACG 703468 CTTCATAAAAGAAAAGATGACA ACG 754235 CATGCCATTGTACTCCAGCC ACG 775821 CCTGCCAGCCTCTAACAGC ACG 775822 TAGTGATGTCTGCTTCAG ACT 775894 TTGCCCAGGCTGGCCTC ACT 775900 GAATGCCAACCTCCCTTCC ACT 775903 TCTGAGTGACAGAGCGA ACT 776251 GCTCCCCGCAACCTCCGC ACG 892088 GCCTCGGCCGCAATCACA ACT 892089 CGGTCACCGTGATGATGGG ACT 892090 AGAATCCCGAGAGATGGTAC CGT 892091 GTCCTTCACCTGAGCTTCC ACT 1036231 GTGTTTACTGTTCAAGGCAAGT ACT 1036232 GGCAACAGAGCAAGACT ACG 1043673 ACTGAGAAACATCATCCCTGGG CGT 1043678 AGTCACAGGCAGTTCACC CGT 1043680 CTGTGACTCCTCTCCTCCCC ACT 1043684 CTGTTTTATACCTGCACAC ACT 1054796 ACGCCAGGCAGGCTCTCA ACT 1059211 CGCCTACTGCCAGAGCAAGCT ACG 1064675 ATTCCTTTTTGCTGAAATAATGAA ACT 1560714 TGGGGCGTGATGGCTCA ACG 1613662 CAACAGAACCACCTTCC ACG 1625609 GTGCACACCTGTAATCC ACG 1625689 CAGGGCTCAAGCGATTCTCC ACG 1626971 TCGCCTGGCCAAAAAAA ACT 1654406 CATTGTACCCCAGGTTGAAAAT CGT 1654409 GTGGTACCACACCCAGCTAATT ACT 1654410 ATCATACTGAGATGCTATCAGAA ACT 1654411 GCACTTTGGGAGGTTGAGG ACT 1654412 CATTCTCCCACCTCAGCCCCC ACG 1654413 CCCGTTTGATTTCCGGGTC CGT 1654415 GGCCCCATCACCCAAAA ACG 1654416 GACCGTCTCATTCACAAAC ACT 1654419 TTGGTGCTTCACTCTGAGAC ACT 1654420 GAGAACCGCTGATCAATGCA CGT 1654421 GCATGCAGCTCCCGTCC ACT 1654424 CCACCTATGGCCGCGCCCCT ACT 1654425 CAGGGACCCATACCTGTGGTC ACT 1654438 TCAGCCTCCTGAGTAGCTGG ACT 1654439 GTTTCTGGAGCACTCCGGT ACT 1654441 GATAAGCCTGGCCAACA ACG 1654442 ATGATCTCGGCTCACTGCAA ACT 1654444 TATGGATCTTTCTAGTCTTGTTT CGT 1654446 ACTGATTACAGGCGTGC ACT 1654447 CCCGATGCCTGTGTTGGC ACT 1654448 AGTGCTGGGATTACAGG ACG 1654449 AATCTCTCTTTTCCCTACACA ACG 1654451 TAATGCGTACAGCAGCC CGT 1654452 ACTGGAGGAGGATGCACTTA ACG 1654459 ATGCACAGAAACAAGGATCTA ACT 1654485 CTTGCTTTTTTTTTTTTTGGACAG ACT 1654491 GCACCCCGAGCCTTTCCAG ACT 1654495 TTGTCGTAAGTCTCTCCTCTCTT CGT 1654496 CGGGAAGGTTGAAGTTGGAC CGT 1654497 CCATTTACAACCAATTGC ACT 1654498 CTTTGTGGGACTTCTTITTTA ACT 1654499 ACCCTGGCCTCCCTAAC ACT 1654503 GGGCAACGTTTGAAGATGCTCTGC ACG 1654504 CACCTTTGACTCTTGAGCC ACT 1654505 TAGCTATGTGCCACCATGCC ACG 1671133 GATTGTAGCTAACTCACAAGG ACT 1671140 TACCTACCCACATCCTATAAAA ACG 1671148 CCTGTAATCCCGGCACT ACT 1671149 CTGGCCACAGTGGCTCA ACG 1671150 CGGGTGACGAAGCCTGAC ACG 1671151 TCCTCTGTGCAAAATCCTCC ACG 1671152 CTCCATCCTGACCCCCGT ACT 1671153 CTGTGGAATTGTGCCTC CGT 1671169 CATGTCCCACAGAGGCTAAC ACT 1671170 GAGAGGGCAATGCCTCAGAG CGT 1671171 TTCTGGGATTCTCTAGAGGG ACT 1671176 AGACATCATCACATCACACCA CGT 1671178 CCAGGCTGGACTCGAACT ACG 1671182 ACTGAATGTCTCGGTATAAAACC ACG 1671187 CAGGTTGAGAAAGCTCTA CGT 1671188 CAGAGTGAGGAGAGTGAGAC ACT 1671191 GAGCGGTTAGAAGATGTGCT ACT 1671192 AAGCCTGTAGGCTTTTAA ACG 1671196 GGGATGACTGAATGAGACAGTA ACG 1671198 CCCTCTTAGCAAATTTCAGCT ACT 1671199 TAACTTTTTTGTGTGTGAGAA ACG 1671214 CGTGCATCCTTCCCACCTA ACG 1671215 GTACTCAAGATGATGTAA CGT 1671216 TTACACCCTGGAGTGGTCC ACT 1671217 TGCCCTCTCGCTGGCTGG ACG 1671218 CAAAGGGAGGTGGTCGCAC ACG 1671219 CAGGAGAATGGTGTGAACC ACG 1671221 AGCTGGGATTACAGGCA ACG 1671223 TACAAAATTAGCTGGGCATG ACT 1671224 CTGTGAGCCGAGATTGC ACT 1671225 CTCAATGTGATCCTCCT ACT 1671226 GCAGGAGAATCACTTGAACTT ACT 1671227 AGATCATGCCATTGCCAGC ACT 1671228 ACAGAAGTTAGCTGGGC ACT 1869616 CTTCAACCTCCGCCTCCTGG ACT 2019599 GAAAAGCATGGGCCGGGCA ACG 2116883 CATACTCACCAATATCTGCT ACT 2124090 GCTTTGTGTTCTTTCTAGTC ACT 2163833 GCCAGCAATGCACGCGCAGT ACG 2217659 GTAACTTACCTAATGATAGAGG ACG 2304167 TGACTCCTTTGGACTGG ACG 2304168 CAGTTCGGTGAAGTGGTT ACT 2365593 GGTGTGAGCCACCACGCC ACG 2365721 AGACTCCCTCTCAAAATAA ACG 2569513 AGGGATAAGCATGAAACCACT ACG 2569514 GCGTGAGCCACCACGCC ACG 2886414 GGGTGACAAAGTGAGACTC ACG 2886415 CACGCCTGGCTAAGCCT ACT 3745912 GGCTGGCAGGCCAGGTCAAC ACT 3786863 GTGCTGGGATTACAGGC ACT Genetic Analysis Allelotyping Results

Allelotyping results are shown for cases and controls in Table 10. The allele frequency for the A2 allele is noted in the fifth and sixth columns for breast cancer pools and control pools, respectively, where “AF” is allele frequency. The allele frequency for the A1 allele can be easily calculated by subtracting the A2 allele frequency from 1 (A1 AF=1−A2 AF). For example, the SNP re269911has the following case and control allele frequencies: case A1 (A)=0.907; case A2 (T)=0.093; control A1 (A)=0.864; and control A2 (T)=0.136, where the nucleotide is provided in paranthesis. SNPs with blank allele frequencies were untyped. TABLE 10 dbSNP Position in Chromosome A2 Case A2 Control rs# Position A1/A2 Allele AF AF p-Value 269911 185 60156885 A/T 0.136 0.093 0.0242 703464 237 60156937 G/A 0.219 0.197 0.3696 703465 641 60157341 C/G 0.608 0.634 0.3828 269912 719 60157419 A/G 0.621 0.651 0.3011 269913 990 60157690 T/C 0.354 0.320 0.2275 269915 2908 60159608 C/T 0.776 0.797 0.3802 269916 3140 60159840 G/A 0.137 0.123 0.5037 172006 3880 60160580 A/G 0.172 0.142 0.1703 703467 4494 60161194 C/T 0.321 0.273 0.0759 703468 5107 60161807 G/A 0.187 0.159 0.2093 2217659 5220 60161920 G/A 0.167 0.127 0.0593 775894 6031 60162731 A/G 0.152 0.131 0.3098 775900 8670 60165370 A/G 0.632 0.667 0.2304 1654491 13794 60170494 T/C 0.005 0.005 0.9971 775903 16356 60173056 A/G 0.818 0.835 0.4460 1036231 17164 60173864 T/C 0.177 0.178 0.9755 1036232 17264 60173964 G/A 0.690 0.713 0.3949 1671223 20537 60177237 T/G 0.951 0.978 0.0143 1671224 20637 60177337 A/G 0.235 0.244 0.7269 1654495 20900 60177600 C/A 0.534 0.536 0.9379 1654496 21155 60177855 C/A 0.315 0.289 0.3567 1654497 21795 60178495 T/C 0.307 0.315 0.7771 1671225 21931 60178631 T/G 0.352 0.344 0.7785 1671226 22167 60178867 C/G 0.520 0.482 0.2147 1671227 22656 60179356 T/C 0.468 0.463 0.8612 1654498 23108 60179808 T/C 0.385 0.387 0.9372 1671228 23404 60180104 T/C 0.412 0.403 0.7790 1654499 24287 60180987 T/C 0.349 0.355 0.8197 1869616 24480 60181180 A/C 0.909 0.925 0.3271 1654503 24592 60181292 C/T 0.384 0.409 0.3829 1654504 24878 60181578 T/C 0.689 0.651 0.1817 1671133 26370 60183070 A/C 0.345 0.348 0.9023 1654505 27056 60183756 G/A 0.257 0.263 0.8407 3786863 27874 60184574 A/G 0.989 0.989 0.9843 1560714 31248 60187948 G/A 0.970 0.981 0.2419 1654406 31458 60188158 G/T 0.437 0.437 0.9994 1043673 31553 60188253 C/A 0.354 0.374 0.4936 1043678 31637 60188337 G/T 0.530 0.525 0.8676 1043680 31668 60188368 C/G 0.544 0.561 0.5849 1043684 31752 60188452 A/G 0.317 0.312 0.8472 1671140 37643 60194343 G/A 0.122 0.116 0.7440 1654409 43941 60200641 A/C 0.557 0.528 0.3425 1654410 44134 60200834 T/C 0.431 0.462 0.3094 1654411 44329 60201029 A/C 0.639 0.578 0.0398 1671148 44343 60201043 A/C 0.859 0.824 0.1102 1671149 44362 60201062 G/A 0.798 0.745 0.0377 1671150 44818 60201518 G/A 0.116 0.148 0.1189 1654412 44917 60201617 C/T 0.371 0.359 0.6627 1671151 45215 60201915 G/A 0.771 0.724 0.0765 1671152 45666 60202366 T/G 0.779 0.720 0.0234 1654413 45680 60202380 T/A 0.186 0.257 0.0048 2304167 46402 60203102 C/T 0.725 0.675 0.0720 1671153 46510 60203210 G/T 0.785 0.700 0.0016 2019599 46554 60203254 C/T 0.051 0.041 0.4200 1654485 46823 60203523 A/C 0.516 0.525 0.7872 1671188 47714 60204414 T/G 0.567 0.595 0.3451 1671191 48963 60205663 T/C 0.131 0.159 0.1931 1654415 49157 60205857 C/T 0.765 0.714 0.0536 1671192 49254 60205954 G/A 0.136 0.180 0.0436 2304168 49257 60205957 A/G 0.200 0.174 0.2713 1654416 49356 60206056 T/C 0.130 0.174 0.0432 1654419 55202 60211902 A/G 0.672 0.627 0.1243 1654420 55527 60212227 T/A 0.704 0.669 0.2206 1613662 55916 60212616 G/A 0.778 0.739 0.1347 2886415 56402 60213102 T/G 0.624 0.561 0.0324 2365593 56413 60213113 C/T 0.131 0.151 0.3369 2886414 56685 60213385 G/A 0.796 0.755 0.0976 1654421 56783 60213483 A/G 0.651 0.616 0.2323 1654424 58044 60214744 A/G 0.056 0.051 0.7154 1654425 58301 60215001 T/C 0.582 0.523 0.0464 892089 58382 60215082 A/G 0.654 0.622 0.2732 892090 58393 60215093 G/T 0.203 0.238 0.1618 1671196 58869 60215569 C/T 0.752 0.699 0.0518 1671198 59155 60215855 T/C 0.037 0.048 0.3599 1671199 59189 60215889 G/A 0.181 0.203 0.3495 1625609 62546 60219246 C/T 0.063 0.063 0.9551 1625689 62568 60219268 G/A Not Alellotyped 1654438 70983 60227683 A/G 0.885 0.890 0.7775 2569513 71465 60228165 G/A 0.777 0.723 0.0373 2569514 71538 60228238 G/A 0.409 0.340 0.0171 1671214 72144 60228844 G/A 0.345 0.392 0.1048 1671215 72340 60229040 C/A 0.596 0.541 0.0649 1054796 72527 60229227 C/G 0.198 0.121 0.0008 1654439 72968 60229668 T/G 0.757 0.712 0.0906 1671216 73397 60230097 A/G 0.239 0.273 0.1934 1671217 73553 60230253 G/A 0.166 0.217 0.0333 1671218 73720 60230420 C/T 0.508 0.450 0.0542 1654441 74190 60230890 C/T 0.893 0.901 0.6470 1654442 74687 60231387 T/G 0.955 0.947 0.5733 1671219 74699 60231399 G/A 0.232 0.217 0.5745 10666 75580 60232280 C/T 0.871 0.879 0.6976 1626971 76345 60233045 T/C 0.099 0.141 0.0329 1671221 76506 60233206 G/A 0.007 0.003 0.3854 754235 77554 60234254 G/A 0.445 0.486 0.1719 775821 77889 60234589 C/T 0.295 0.310 0.5716 3745912 77919 60234619 A/G 0.730 0.736 0.8182 775822 78866 60235566 A/G 0.983 0.991 0.2487 1059211 79061 60235761 C/T 0.231 0.205 0.2919 2124090 83777 60240477 A/C 0.681 0.772 0.0010 1671171 84360 60241060 T/G 0.222 0.259 0.1467 1671170 84631 60241331 T/A 0.546 0.517 0.3441 1654444 85775 60242475 G/T 0.170 0.208 0.1062 2365721 87153 60243853 G/A 0.443 0.409 0.2552 1654446 89650 60246350 A/G 0.430 0.388 0.1582 1654447 89895 60246595 A/G 0.647 0.624 0.4372 1671176 90103 60246803 C/A 0.246 0.274 0.2919 1654448 90234 60246934 G/A 0.136 0.164 0.1947 1671178 90309 60247009 G/A 0.037 0.036 0.9262 1654449 90376 60247076 G/A 0.167 0.186 0.4301 1671182 90925 60247625 C/T 0.211 0.207 0.8737 1654451 91561 60248261 A/T 0.734 0.691 0.1110 1654452 91605 60248305 G/A 0.322 0.351 0.3085 1671169 92954 60249654 T/C 0.532 0.554 0.4612 1654459 94228 60250928 A/G 0.137 0.158 0.3262 269909 Not Mapped G/C 0.777 0.799 0.3619 269910 Not Mapped T/G 0.234 0.190 0.0702 776251 Not Mapped G/A 0.013 0.008 0.3916 892088 Not Mapped A/G 0.540 0.523 0.5742 892091 Not Mapped C/G 0.471 0.478 0.8145 1043680 Not Mapped C/G 0.544 0.561 0.5849 1064675 Not Mapped A/G 0.193 0.210 0.4668 1671187 Not Mapped T/A 0.607 0.594 0.6661 2116883 Not Mapped T/C 0.177 0.227 0.0409 2163833 Not Mapped G/A 0.156 0.209 0.0227

FIG. 14 shows the proximal SNPs in and around the GP6 gene. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group. The minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in FIG. 14 can be determined by consulting Table 10. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.

To aid the interpretation, multiple lines have been added to the graph. The broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01. The vertical broken lines are drawn every 20 kb at assist in the interpretation of distances between SNPs. Two other lines are drawn to expose linear trends in the association of SNPs to the disease. The light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W. S. Cleveland, E. Grosse and W. M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J. M. Chambers and T. J. Hastie, Wadsworth & Brooks/Cole.). The black line (or generally top-most curve, e.g., see peak in left-most graph just to the left of position 92150000) provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10 kb sliding window with 1 kb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10⁻⁸ were truncated at that value.

Finally, the gene or genes present in the loci region of the proximal SNPs as annotated by Locus Link (http address: www.ncbi.nlm.nih.gov/LocusLink/) are provided on the graph. The exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3′ end of each gene to show the direction of transcription.

Additional Genotyping

In addition to the SNP rs1671152, another SNP (rs1654416) was genotyped in the discovery cohort and found to be associated with breast cancer with a p-value of 0.0737. See Table 13.

The methods used to verify and genotype the proximal SNP of Table 13 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 11 and Table 12, respectively. TABLE 11 dbSNP First Second rs# PCR primer PCR primer 1654416 ACGTTGGATGTCTGCTGAGCATGAAATGCC ACGTTGGATGCTGAACTGACCGTCTCATTC

TABLE 12 dbSNP Extend Term rS# Primer Mix 1654416 TGACCGTCTCATTCACAAAC ACT

Table 13, below, shows the case and control allele frequencies along with the p-values for the SNPs genotyped. The disease associated allele of column 4 is in bold and the disease associated amino acid of column 5 is also in bold. The chromosome position provided corresponds to NCBI's Build 33. TABLE 13 Genotpying Results Amino dbSNP Position in Chromosome Alleles Acid AF AF Odds rs# Position (A1/A2) Change F case F control p-value Ratio 1654416 49356 60206056 T/C E237K T = 0.840 T = 0.800 0.0737 0.75 C = 0.160 C = 0.200

Example 4 LAMA4 Proximal SNPs

It has been discovered that a polymorphic variation (FCH-1159) in a region that encodes laminin, alpha (LAMA4) is associated with the occurrence of breast cancer (see Examples 1 and 2). Subsequently, SNPs proximal to the incident SNP (FCH-1159) were identified and allelotyped in breast cancer sample sets and control sample sets as described in Examples 1 and 2. Approximately sixty-eight allelic variants located within the LAMA4 region were identified and allelotyped. The polymorphic variants are set forth in Table 14. The chromosome position provided in column four of Table 14 is based on Genome “Build 33” of NCBI's GenBank. TABLE 14 dbSNP Position in Chromosome Allele rs# Chromosome Position Variants 969138 6 184 112446684 C/G 1418499 6 506 112447006 T/C 2157550 6 3981 112450481 C/G 764587 6 7815 112454315 A/G 3734287 6 7875 112454375 G/A 2032565 6 10775 112457275 T/A 2032566 6 10786 112457286 T/C 1050349 6 11013 112457513 G/C LAMA4_SNP1 6 11020 112457520 C/T 2032568 6 11101 112457601 A/G 2072019 6 14171 112460671 A/G 2072020 6 14278 112460778 G/A 1480646 6 16512 112463012 G/A 2072026 6 16706 112463206 C/T 763247 6 18442 112464942 A/C 744006 6 20286 112466786 C/T 3822941 6 21591 112468091 G/A 3777942 6 22275 112468775 A/T 3734286 6 25318 112471818 C/G 3777941 6 27997 112474497 C/T 2277084 6 29840 112476340 A/G 3798359 6 31088 112477588 C/G 3798357 6 31258 112477758 T/C 2227237 6 32367 112478867 G/A 2213838 6 32427 112478927 T/C 3752577 6 33671 112480171 G/A LAMA4_SNP2 6 38796 112485296 C/T 971402 6 41530 112488030 A/G 971405 6 41874 112488374 A/T 2051649 6 44161 112490661 A/G 1050348 6 47502 112494002 C/T 2157544 6 51089 112497589 T/G 2157545 6 51205 112497705 C/T 2213839 6 53645 112500145 G/A LAMA4_SNP4 6 54280 112500780 G/T 3777934 6 57610 112504110 A/G 1894681 6 57740 112504240 A/G 2301512 6 60812 112507312 G/C 2301513 6 60837 112507337 T/C 2072029 6 64448 112510948 T/C 764071 6 65249 112511749 T/G 2269646 6 65482 112511982 T/C LAMA4_SNP5 6 66535 112513035 C/T 2072022 6 66789 112513289 T/C LAMA4_SNP6 6 67214 112513714 C/A 2237238 6 68347 112514847 C/A 3777932 6 69060 112515560 T/C 3777929 6 70100 112516600 A/G 3777928 6 70215 112516715 G/T 2157546 6 73687 112520187 C/G 3948760 6 73732 112520232 A/G 2237241 6 74183 112520683 T/C 2237242 6 74813 112521313 A/G 2237244 6 78136 112524636 T/C 3777927 6 79540 112526040 C/T 3777926 6 79655 112526155 A/G 3777925 6 79731 112526231 T/C 2239849 6 82111 112528611 G/A 2239850 6 82155 112528655 T/G 2237247 6 83479 112529979 G/A 2282853 6 84511 112531011 G/C 2282854 6 85290 112531790 T/C 2213840 6 90620 112537120 G/A 2068770 6 91127 112537627 A/G 2237248 6 92095 112538595 G/A 2237249 6 92679 112539179 A/G 2345808 6 94839 112541339 T/G 2157547 6 95220 112541720 G/C

Assay for Verifying and Allelotyping SNPs

The methods used to verify and allelotype the proximal SNPs of Table 14 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 15 and Table 16, respectively. The methods used to verify and allelotype the proximal SNPs of Table 14 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 15 and Table 16, respectively. TABLE 15 dbSNP Forward Reverse rs# PCR primer PCR primer LAMA4_SNP5 ACGTTGGATGACAGTTCTTGGCTATCCTGG ACGTTGGATGACTGGCCAGTGTAGGAATTG LAMA4_SNP6 ACGTTGGATGGAAAGGGATTGACTCAGGAG ACGTTGGATGCTTCCTTCACCTGAAGATGG LAMA4_SNP4 ACGTTGGATGTTGAAGGACTGATCTATGGG ACGTTGGATGAAAGCAACAGACAAGGCAAG LAMA4_SNP1 ACGTTGGATGCAGACTGGAAATGCGCAATG ACGTTGGATGCGTATCTTCAAGATGCACAG 1050348 ACGTTGGATGTGTTCATGTCTTCGGCATCC ACGTTGGATGCAGCTGGATGACTACAATGC LAMA4_SNP2 ACGTTGGATGAGGAATGCTTACAACGGAGG ACGTTGGATGAACTCCCTTCATCCTTCCTC  744006 ACGTTGGATGTTGCCTTGAAGGTAGGCATG ACGTTGGATGGGGTTAGCAGCTTAACTTTC  763247 ACGTTGGATGCCGGCCAAGACCAATACATC ACGTTGGATGTGCAGACATGCACTATTCTC  764071 ACGTTGGATGCCACTTGGAAAGATTCAAGG ACGTTGGATGTATTGTGACTTCTGCAGAAC  764587 ACGTTGGATGCAACATAGACCAGAAGTGGG ACGTTGGATGTTCACATAGGGAAGGCCTTG  969138 ACGTTGGATGACTGGACCAAGGTAGATCAC ACGTTGGATGCTCAGGCTAATCTCTCTAGG  971402 ACGTTGGATGCCACTTTTCTGTGGAAATATC ACGTTTGGATGCAAGTTAATGAGTTTCTCCC  971405 ACGTTGGATGAAACAGTGCTTTTTGAAGGAG ACGTTGGATGCTATCTCCAAAGGGTAACAG 1050348 ACGTTGGATGCAGCTGGATGACTACAATGC ACGTTGGATGTGTTCATGTCTTCGGCATCC 1050349 ACGTTGGATGCTATGATTTTGGATTCAGCG ACGTTGGATGACCTCATGGTATTTTGCATC 1158747 ACGTTGGATGTTGAAGGACTGATCTATGGG ACGTTGGATGAAAGCAACAGACAAGGCAAG 1418499 ACGTTGGATGACCATAGGGAACTAGAAATC ACGTTGGATGCTTTAAGATAGATTCCCAGGG 1480646 ACGTTGGATGCAGTGTCTCTTCCTTTCCAG ACGTTGGATGCAAATTTCCACGAGCCTGAG 1894681 ACGTTGGATGTGGGATTCCCCTAAAGGATG ACGTTGGATGAAGATCAGCAGCACCAAAGG 2032565 ACGTTGGATGAAAGAGCAACTGAAGGACCC ACGTTGGATGTAAATTGGAACATCAACAGG 2032566 ACGTTGGATGTAAATTGGAACATCAACAGG ACGTTGGATGAAAGAGCAACTGAAGGACCC 2032567 ACGTTGGATGCGTATCTTTCAAGATGCACAG ACGTTGGATGAGACTGGAAATGCGCAATGG 2032568 ACGTTGGATGACTCGCATAACAGATGTTCC ACGTTGGATGTAACCATTGCGCATTTCCAG 2051649 ACGTTGGATGACCTGCTGAAAACCAACACC ACGTTGGATGGGAGAGGAGAACCCTGGAC 2068770 ACGTTGGATGCACTTCACGTACTTCACTGG ACGTTGGATGAGTTTGCTCCTATGTGGCTC 2072019 ACGTTGGATGAGGTCCACAGAAGATGTTAG ACGTTGGATGCACAACGGTCATTTGAACAC 2072020 ACGTTGGATGAAGTCCTGTTGTCTGCAAGG ACGTTGGATGCAGTTGTCTTAGCACACAGG 2072022 ACGTTGGATGCAAAGAAGAAAGATGTAGTGG ACGTTGGATGCGAAATCTGGTCCTATGAAG 2072026 ACGTTGGATGTCCTATCACCATCACACTAC ACGTTGGATGCAGCATCAAACAGAATAGGC 2072029 ACGTTGGATGTCCTTTGCAGACTGATACTCC ACGTTGGATGTCACTCACTCCTTGCTAAGC 2157544 ACGTTGGATGCATATGTAGTAGGAATGAGGG ACGTTGGATGTGAGGCTCAAAGGGATTAGG 2157545 ACGTTGGATGTCTGGTCAACCACATAGATC ACGTTGGATGTGTTCTACTGCAGCTCCAAG 2157546 ACGTTGGATGTCCACTTGTACAGAATGGAG ACGTTGGATGCATTTACTCAGTGCCAGGTC 2157547 ACGTTGGATGCCATACCATTTACTTCTGCC ACGTTGGATGAGGCAAGTACACATACAATG 2157550 ACGTTGGATGCACACACACATTTTAATTGCC ACGTTGGATGTTGTTCAGAATTACATGATG 2213838 ACGTTGGATGAAAGGACTTGAGGGTGATTG ACGTTGGATGGCAACAAACAGTGTTCCAGC 2213839 ACGTTGGATGAGTCACAGTTCAGTCCCAAC ACGTTGGATGGGGCAATTTTCTAGTCCAGC 2213840 ACGTTGGATGCTTTCGCACAAGGCTCTATC ACGTTGGATGAAGTCTGTGTTTAAGCCCCC 2227237 ACGTTGGATGGATGTCTCTAAGTTGAAATGC ACGTTGGATGATATCAATCACCCTCAAGTC 2237238 ACGTTGGATGCAGAGGCTGAAGGAACATAC ACGTTGGATGTCTGTAATCCCAGGACCCTA 2237241 ACGTTGGATGTCAGCAGGGCTCTATCTAAG ACGTTGGATGCCAAGCAGTATTGCTAATGG 2237242 ACGTTGGATGCCTCACCATTGTGTTTAGGC ACGTTGGATGTGACTATTTCCGCTTGGCTC 2237244 ACGTTGGATGGAGAAAAATAGACTCGGCCC ACGTTGGATGCACAGACGCAGGATTTGGAT 2237247 ACGTTGGATGCTGCTTCTCCAGTAATGTTG ACGTTGGATGGTGTTAGTAACACTGATGCC 2237248 ACGTTGGATGCCCTCCCCAGATATCATTAG ACGTTGGATGCATATCCACAGCCTAATCAC 2237249 ACGTTGGATGAAATGCTTCCTACTGCAATC ACGTTGGATGTGGAGAGTTGTGGTTGATGG 2239849 ACGTTGGATGGACATCAGATCAGACAGCAC ACGTTGGATGACTTTCTGGCATTGACTGGG 2239850 ACGTTGGATGGCCCAGGAAAAATTAATTCAC ACGTTGGATGGCAGTACGGATTAGCATGAG 2269646 ACGTTGGATGTCACCTCACTTTTGAAGAGC ACGTTGGATGTCTGGTTAGGCTTCAGTTAG 2277084 ACGTTGGATGGAGGGTAAAAATGACAGCAG ACGTTGGATGTTTTGCTTGGTGTTTAGCAG 2282853 ACGTTGGATGTCTTGACCTTCCTGGTTTTC ACGTTGGATGTATCAGAGCTAGAAGAAACC 2282854 ACGTTGGATGTAGCCAGTGGTTAAGAAAGC ACGTTGGATGTTCTCATGTTGGGGAGACAC 2301512 ACGTTGGATGATCTGAGTGGTTTCAGGAGG ACGTTGGATGACCTGTTGGAACACATGAAG 2301513 ACGTTGGATGCTGGCGGGTAGTGTCTTCAT ACGTTGGATGCTTTGAAATTGTTCTTGTCC 2345808 ACGTTGGATGTTCTGGGATTTAAAGGAGGC ACGTTGGATGCCAAACATTTCTTGTTGGAC 3734286 ACGTTGGATGACCTTACACTCCAGTGAATC ACGTTGGATGGCCGTTAAGCAACTACAAGC 3734287 ACGTTGGATGCAGTGGAGAAGATGAAACCC ACGTTGGATGGCCACTTCTGGTCTATGTTG 3752577 ACGTTGGATGCATGGCTGAGGTTACTTAGG ACGTTGGATGGAATGCGTCAGGGATTTATG 3777925 ACGTTGGATGCTACAAGTCTAACAGTCAGAG ACGTTGGATGTTTACAGAGCAAGGTCTGAGG 3777926 ACGTTGGATGGTGAGTACCATCCCTTTTGC ACGTTGGATGCTGTTAAACTGCCTCAGACC 3777927 ACGTTGGATGAAACGAATGCTTGAGAGCAG ACGTTGGATGGTCCTGATTTATGAGCTCCC 3777928 ACGTTGGATGTTCACACGTAGACCCTGTTG ACGTTGGATGTCAGGAGTTGAGCAAGCTAG 3777929 ACGTTGGATGGCTGTCTTTGGGATTAAAT ACGTTGGATGTTCATAAAGAAGTGGAGAGC 3777932 ACGTTGGATGTCCCAGACCTTAAGATTCCC ACGTTGGATGTATTAGGCTCTTTGGCCGAC 3777934 ACGTTGGATGCAAGATCCAGATGGTGAGGG ACGTTGGATGCAAGGTCAGAGTGTCACTGG 3777941 ACGTTGGATGGCTTCCTGAGATTATATTGAC ACGTTGGATGCTCCATTCCAAATTCCTTTC 3777942 ACGTTGGATGTCATGACAAATCATGACTAG ACGTTGGATGTCAGATACAAGTGAAGGTAG 3798357 ACGTTGGATGTCCCAATTCAGGAAATGGTG ACGTTGGATGTGCTTGGTATACCATGCCTG 3798359 ACGTTGGATGTTCCTCAGCACACAGCCCCA ACGTTGGATGATGAACCTTACACAGGCCAG 3822941 ACGTTGGATGTATAATAAACTGATAGTTGC ACGTTGGATGCTCTGTACTTAGGACACACG 3948760 ACGTTGGATGTCCACTTGTACAGAATGGAG ACGTTGGATGTCCCACACTCAAAACTTTGC

TABLE 16 dbSNP Extend Term rs# Primer Mix LAMA4_SNP5 ATTGCTTTACGCAACACCAC ACG LAMA4_SNP6 AGATGGAGAGAATGCCAC CGT LAMA4_SNP4 GCAAGTGGGCATTCGACCA CGT LAMA4_SNP1 CTTCAAGATGCACAGGGCCAC ACG 1050348 CACTTGACCAGGCCCTTAAC ACG LAMA4_SNP2 GGCCCGCCTGCATCTGTG ACG  744006 CTTTCTCTCTTTCCAGG ACG  763247 CTTTTAATCCCCCACACT ACT  764071 AGAACATATATGTTGCATTTTTTT ACT  764587 GAAGGCCTTTGCCTGTTA ACT  969138 AGGAAGAGAATCTGATAGCC ACT  971402 AGTTTCTCCCACTTACC ACT  971405 AGGGTAACAGAATGATTAAAA CGT 1050348 TTCGGCATCCCTGACAT ACT 1050349 CGTATCTTCAAGATGCACA ACT 1158747 GCAAGTGGGCATTCGACCA CGT 1418499 GGGCAGAATTACTGAATCAAG ACT 1480646 CAGCAGACTCTGATGTGGC ACG 1894681 GGGAGCATCTTTTGAGC ACT 2032565 CAACAGGAAAAATACATCCA CGT 2032566 CAACCCTAGGAAAACATTT ACT 2032567 TTCTATGATTTTGGATTCAGC ACT 2032568 ACATACTCTGAGGAGAGAAAG ACT 2051649 GAACCCTGGACAAGAAT ACT 2068770 ATGTGGCTCAAACATCCGAA ACT 2072019 TTTGAACACTACAGTTTCTGTTAT ACT 2072020 AAACAATCCATTTAACATACCTA ACG 2072022 GCAAATGAATTCTGGGA ACT 2072026 TGAAAGTCTTTGAGGTGTT ACG 2072029 CCTGGCAATGATCAACCCCC ACT 2157544 CTAAATATTAGCAGACTGAAATAC ACT 2157545 GCTGGCATAAATGAAATTG ACG 2157546 GTGCCAGGTCCCACACT ACT 2157547 GTACACATACAATGATTTTACTC ACT 2157550 TTACATGATGAATATTATGGAAGT ACT 2213838 TTCCAGCATGATTCTAAGACA ACT 2213839 CAACTTGAGATACAGTAAAAATT ACG 2213840 TGAAATGAATTCTCCAATAGAC ACG 2227237 ACCCTCAAGTCCTTTTG ACG 2237238 TCCCAGGACCCTAAAAAAGT CGT 2237241 CAGTATTGCTAATGGGTGTTC ACT 2237242 TGTCTCTAGGGCACTACATATC ACT 2237244 GAAATAATGCTTCAGGGG ACT 2237247 ATGCCTTCTAATGCATTCATTTTA ACG 2237248 CCTAATCACATAAACCAGGAA ACG 2237249 GAAAACAAGAGAGGGAAG ACT 2239849 TGTGACTCCTCATGCTAATC ACG 2239850 CCAGTCAATGCCAGAAA ACT 2269646 CAGTTAGACTGAAACGCACA ACT 2277084 TGTGTCATTTAAATCCTTCA ACT 2282853 GAGCTAGAAGAAACCTGAAAG ACT 2282854 GTTGGTGTCCAAATGGCA ACT 2301512 ACATGAAGACACTACCC ACT 2301513 TGTTCTTGTCCAAAATTACCT ACT 2345808 GACATTTAGGTTATTTCCAAATTT ACT 3734286 CATCAGAGAGAATTGAAGT ACT 3734287 GGAATTCAGGCATACAC ACG 3752577 AGAAATAGATGGAGCCAAAAG ACG 3777925 GGATGGGACTGAAACTC ACT 3777926 CTCTGTAATTTTTCATGTATGATA ACT 3777927 ATGAGCTCCCTTCACTC ACG 3777928 TGAGCAAGCTAGAGAGTA CGT 3777929 AAGTGGAGAGCATTTACAT ACT 3777932 TTTGGCCGACTGAAATG ACT 3777934 GGTCAGAGTGTCACTGGGCTACA ACT 3777941 TCCCAAATTTCCTTTTCA ACG 3777942 ACAAGTGAAGGTAGTATTGT CGT 3798357 GCCTGGCATCTGCTAATC ACT 3798359 GCAAAGGCAGAGACTAT ACT 3822941 ACACACGATGTTTCTCCAG ACG 3948760 ACAGTTTTATGAGACAGGTA ACT Genetic Analysis and Allelotyping Results

Allelotyping results are shown for cases and controls in Table 17. The allele frequency for the A2 allele is noted in the fifth and sixth columns for breast cancer pools and control pools, respectively, where “AF” is allele frequency. The allele frequency for the Al allele can be easily calculated by subtracting the A2 allele frequency from 1 (A1 AF=1−A2 AF). For example, the SNP rs969138 has the following case and control allele frequencies: case A1 (C)=0.893; case A2 (G)=0.107; control A1 (C)=0.866; and control A2 (G)=0.134, where the nucleotide is provided in paranthesis. SNPs with blank allele frequencies were untyped. TABLE 17 dbSNP Position in Chromosome A1/A2 A2 Case A2 Control rs# Position Allele AF AF p-Value  969138 184 112446684 C/G 0.107 0.134 0.1800 1418499 506 112447006 T/C 0.474 0.545 0.0181 2157550 3981 112450481 C/G 0.238 0.294 0.0356  764587 7815 112454315 A/G 0.263 0.192 0.0054 3734287 7875 112454375 G/A 0.645 0.729 0.0032 2032565 10775 112457275 T/A 0.271 0.303 0.2393 2032566 10786 112457286 T/C 0.560 0.527 0.2791 1050349 11013 112457513 G/C 0.789 0.806 0.4918 LAMA4_SNP1 11020 112457520 C/T 0.556 0.480 0.0109 2032568 11101 112457601 A/G 0.280 0.336 0.0429 2072019 14171 112460671 A/G 0.658 0.608 0.0830 2072020 14278 112460778 G/A 0.171 0.170 0.9646 1480646 16512 112463012 G/A 0.329 0.329 0.9962 2072026 16706 112463206 C/T 0.326 0.367 0.1525  763247 18442 112464942 A/C 0.293 0.360 0.0168  744006 20286 112466786 C/T 0.697 0.670 0.3444 3822941 21591 112468091 G/A 0.694 0.670 0.3923 3777942 22275 112468775 A/T 0.234 0.254 0.4484 3734286 25318 112471818 C/G 0.240 0.249 0.7505 3777941 27997 112474497 C/T 0.590 0.573 0.5688 2277084 29840 112476340 A/G 0.170 0.193 0.3128 3798359 31088 112477588 C/G 0.189 0.194 0.8418 3798357 31258 112477758 T/C 0.607 0.589 0.5393 2227237 32367 112478867 G/A 0.699 0.678 0.4600 2213838 32427 112478927 T/C 0.619 0.606 0.6603 3752577 33671 112480171 G/A 0.866 0.856 0.6307 LAMA4_SNP2 38796 112485296 C/T 0.480 0.441 0.1869  971402 41530 112488030 A/G 0.406 0.397 0.7632  971405 41874 112488374 A/T 0.396 0.370 0.3662 2051649 44161 112490661 A/G 0.620 0.596 0.4216 1050348 47502 112494002 C/T 0.437 0.518 0.0071 2157544 51089 112497589 T/G 0.344 0.396 0.0695 2157545 51205 112497705 C/T 0.143 0.116 0.1789 2213839 53645 112500145 G/A 0.329 0.387 0.0415 LAMA4_SNP4 54280 112500780 G/T 0.339 0.392 0.0645 3777934 57610 112504110 A/G 0.065 0.068 0.8646 1894681 57740 112504240 A/G 0.433 0.382 0.0840 2301512 60812 112507312 G/C 0.089 0.092 0.8857 2301513 60837 112507337 T/C 0.688 0.741 0.0514 2072029 64448 112510948 T/C 0.765 0.782 0.5146  764071 65249 112511749 T/G 0.431 0.502 0.0175 2269646 65482 112511982 T/C 0.438 0.379 0.0458 LAMA4_SNP5 66535 112513035 C/T 0.099 0.071 0.1061 2072022 66789 112513289 T/C 0.956 0.954 0.9004 LAMA4_SNP6 67214 112513714 C/A 0.410 0.452 0.1649 2237238 68347 112514847 C/A 0.111 0.099 0.5343 3777932 69060 112515560 T/C 0.080 0.073 0.6848 3777929 70100 112516600 A/G 0.311 0.364 0.0656 3777928 70215 112516715 G/T 0.414 0.364 0.0861 2157546 73687 112520187 C/G 0.331 0.415 0.0045 3948760 73732 112520232 A/G 0.187 0.269 0.0015 2237241 74183 112520683 T/C 0.384 0.447 0.0360 2237242 74813 112521313 A/G 0.434 0.391 0.1478 2237244 78136 112524636 T/C 0.492 0.515 0.4374 3777927 79540 112526040 C/T 0.110 0.042 0.0001 3777926 79655 112526155 A/G 0.854 0.858 0.8519 3777925 79731 112526231 T/C 0.774 0.771 0.9216 2239849 82111 112528611 G/A 0.734 0.771 0.1596 2239850 82155 112528655 T/G 0.851 0.936 0.0000 2237247 83479 112529979 G/A 0.097 0.093 0.8578 2282853 84511 112531011 G/C 0.775 0.783 0.7522 2282854 85290 112531790 T/C 0.078 0.014 0.0000 2213840 90620 112537120 G/A 0.738 0.806 0.0077 2068770 91127 112537627 A/G 0.332 0.277 0.0479 2237248 92095 112538595 G/A 0.212 0.153 0.0111 2237249 92679 112539179 A/G 0.339 0.270 0.0128 2345808 94839 112541339 T/G 0.182 0.114 0.0019 2157547 95220 112541720 G/C 0.176 0.128 0.0258

FIG. 15 shows the proximal SNPs in and around the LAMA4 region. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group. The minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in FIG. 15 can be determined by consulting Table 17. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.

To aid the interpretation, multiple lines have been added to the graph. The broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01. The vertical broken lines are drawn every 20 kb to assist in the interpretation of distances between SNPs. Two other lines are drawn to expose linear trends in the association of SNPs to the disease. The light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W. S. Cleveland, E. Grosse and W. M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J. M. Chambers and T. J. Hastie, Wadsworth & Brooks/Cole.). The black line (or generally top-most curve, e.g., see peak in left-most graph just to the left of position 92150000) provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10 kb sliding window with 1 kb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10⁻⁸ were truncated at that value.

Finally, the gene or genes present in the loci region of the proximal SNPs as annotated by Locus Link (http address: www.ncbi.nlm.nih.gov/LocusLink/) are provided on the graph. The exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3′ end of each gene to show the direction of transcription.

Example 5 CHGB/C20orf154 Proximal SNPs

It has been discovered that a polymorphic variation (rs454422) in a gene region that includes CHGB and C20orf154 is associated with the occurrence of breast cancer (see Examples 1 and 2). Subsequently, SNPs proximal to the incident SNP (rs454422) were identified and allelotyped in breast cancer sample sets and control sample sets as described in Examples 1 and 2. A total of ninety-eight allelic variants located within or nearby the CHGB/C20orf154 region were identified and allelotyped. The polymorphic variants are set forth in Table 18. The chromosome position provided in column four of Table 18 is based on Genome “Build 33” of NCBI's GenBank. TABLE 18 dbSNP Position in Chromosome Allele rs# Chromosome Position Variants 2300427 20 186 5842586 T/G 571039 20 1332 5843732 G/T 236143 20 1893 5844293 T/C 236145 20 2786 5845186 C/G 236146 20 2962 5845362 A/G 446658 20 3377 5845777 A/C 500277 20 5522 5847922 A/G 2268339 20 5621 5848021 A/G 454328 20 5889 5848289 G/A 236148 20 7531 5849931 T/C 236149 20 8268 5850668 T/C 910122 20 8923 5851323 G/A 881118 20 8988 5851388 A/C 236151 20 9117 5851517 G/A 236152 20 9448 5851848 C/G CHGB_SNP1 20 9494 5851894 G/A 742710 20 9628 5852028 G/A 742711 20 9640 5852040 T/C 236154 20 11072 5853472 T/G 236155 20 11150 5853550 A/G 54144 20 11379 5853779 C/A 540717 20 11692 5854092 G/A 236158 20 12056 5854456 T/C 236159 20 12104 5854504 G/A 446614 20 14160 5856560 T/C 1343180 20 14836 5857236 A/G 1039542 20 14980 5857380 A/C 400735 20 15165 5857565 A/G 1039543 20 15315 5857715 A/G 440005 20 15624 5858024 A/C 394604 20 15796 5858196 C/T 546106 20 15939 5858339 T/C 452749 20 16581 5858981 C/T 364652 20 17045 5859445 T/C 403727 20 18501 5860901 A/G 236160 20 21800 5864200 A/G 236161 20 21966 5864366 A/G 236162 20 22134 5864534 A/G 236163 20 22181 5864581 A/G 236164 20 23028 5865428 A/G 236165 20 23312 5865712 A/G 236166 20 23573 5865973 T/A 236167 20 23858 5866258 A/G 183535 20 23888 5866288 G/A 236168 20 23990 5866390 T/C 236169 20 24073 5866473 A/G 236171 20 25330 5867730 G/A 236173 20 26473 5868873 T/C 236175 20 27958 5870358 C/T 236176 20 28421 5870821 A/G 451571 20 28804 5871204 C/T 236177 20 29322 5871722 C/A 1005517 20 30819 5873219 G/C 236179 20 31956 5874356 G/A 236180 20 32592 5874992 G/A 236181 20 32818 5875218 G/C 236182 20 32880 5875280 G/T 236183 20 33244 5875644 G/C 236184 20 33845 5876245 A/G 236185 20 34272 5876672 G/A 236187 20 34931 5877331 T/C 1394095 20 36870 5879270 T/G 236189 20 37790 5880190 T/C 236110 20 38708 5881108 T/G 236111 20 39135 5881535 T/C 236112 20 39919 5882319 A/G 236113 20 40166 5882566 C/T 236114 20 40985 5883385 A/G 236115 20 41049 5883449 A/G 236116 20 41935 5884335 C/T 236117 20 42775 5885175 A/C 236118 20 43807 5886207 T/C 236119 20 44254 5886654 A/G 3761873 20 44814 5887214 A/C 236120 20 45249 5887649 T/G 451417 20 47599 5889999 C/A 379418 20 47807 5890207 G/A 2326680 20 48555 5890955 A/C 409035 20 49249 5891649 G/A 454422 20 49293 5891693 A/C 236102 20 57566 5899966 A/C 236103 20 63587 5905987 T/C 236104 20 64560 5906960 C/T 236105 20 65432 5907832 C/G 236106 20 66291 5908691 T/C 236107 20 71331 5913731 A/T 180477 20 73344 5915744 A/T 236108 20 74159 5916559 C/T 236109 20 74564 5916964 T/C 236121 20 78194 5920594 A/G 236122 20 79128 5921528 T/C 236123 20 79393 5921793 C/T 236124 20 81579 5923979 G/A 236125 20 82574 5924974 C/T 2876003 20 85309 5927709 G/C CHGB_SNP2 20 87076 5929476 A/G 2423131 20 87844 5930244 C/A 2206817 20 90241 5932641 T/C Assay for Verifying and Allelotyping SNPs

The methods used to verify and allelotype the sixty-three proximal SNPs of Table 18 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 19 and Table 20, respectively. TABLE 19 dbSNP Forward Reverse rs# PCR primer PCR primer    5414 AACGTTGGATGAGCAGCCATCACATGATCTG ACGTTGGATGCCAATTGTGTCAAACATTTAATGAA  183535 ACGTTGGATGTGGTCTAAGTCCTGCACAAG ACGTTGGATGGACAGAAAGAGTGTGCAGTC  236104 ACGTTGGATGCTCCCTGAACTTCCCATTTC ACGTTGGATGCAGGGAGCTTACTACAAAGC  236105 ACGTTGGATGGGTAGCCACATTTGACACAG ACGTTGGATGAAGTGGCCTGGAAACCAATG  236113 ACGTTGGATGGCTAATACATCCTAAAGGAAC ACGTTGGATGCTTTATTGGAAATCTGTTCG  236119 ACGTTGGATGTTTCTTCTTGTCTCACAGGC ACGTTGGATGCTGTAGATTTTCCTTTTGGC  236120 ACGTTGGATGCAGGATAATGATCATCCCTG ACGTTGGATGACTGGCACAATTAGTGTCTG  236122 ACGTTGGATGTAGCTATCCCATTGTTGAGG ACGTTGGATGGCTAAAGTCTGAAAACACTAG  236143 ACGTTGGATGCCTACCCCAAATAGGTAAAG ACGTTGGATGAGGAAGTAAGTTTTGGGAGG  236145 ACGTTGGATGAGAATGTGAGCAAGGGATGC ACGTTGGATGAATGGCACAGCTATCCTCAG  236146 ACGTTGGATGGTAAATCTGTATCTCCGCCC ACGTTGGATGGACTTGATTACGTGACCTGG  236149 ACGTTGGATGCCAATGATCAAACTGAAATCG ACGTTGGATGGGACTGGATTAGATGAATTC  236160 ACGTTGGATGCACCCACAGCTATCTGAGTT ACGTTGGATGTCCAAAAGGAGTGGGTAGAG  236162 ACGTTGGATGAATGGAGTGTTTGCATGTTG ACGTTGGATGCATGGATTTTTAGGACATGCG  236163 ACGTTGGATGGCCAAAAATAGCCTTTTCTC ACGTTGGATGAAACAACATGCAAACACTCC  236164 ACGTTGGATGTTGCAAGCTGGTGTCACACA ACGTTGGATGAGACCAGCCACTACTGATTC  236166 ACGTTGGATGCTCCTGTCATAGAATAGGCC ACGTTGGATGGCATAGCACATGCTATTTGG  236167 ACGTTGGATGATCAAAGTCTTGTGCAGGAC ACGTTGGATGGCACTTTAGGGACATTTTGAC  236176 ACGTTGGATGGCATAGATAGCTTAATCATGG ACGTTGGATGAAACCAATAGAAGCAGGTTG  236177 ACGTTGGATGCATTTGAGGATCGGAGTGAG ACGTTGGATGCCCTGTGTCTGCAAATTTGG  236180 ACGTTGGATGCCTAGCACTTGGGAATTAGG ACGTTGGATGGATGCGTGAAATAGATGCTC  236182 ACGTTGGATGCTCTCTAGTTCCTTTGTTGC ACGTTGGATGATTTCAAAAGTGGTCTCCAC  236183 ACGTTGGATGTAAAAGAGAAATCCCACAGG ACGTTGGATGCTCAGCAGATGTTAGTTTTC  236184 ACGTTGGATGCTAGGGACCCAGCAAATAAC ACGTTGGATGACCATCTGAGGGAAATCCTG  379418 ACGTTGGATGTCTGGCCTCTAAGCTAAAGG ACGTTGGATGCAGTGGTGTTGATAGATGGG  400735 ACGTTGGATGACCTTCTCAGGTTCACGTTC ACGTTTGGATGAAGGGCACCATGCCATTAAC  409035 ACGTTGGATGTGCTATGGGTATGCAAGGTG ACGTTGGATGGGTTGTTGAAGGAGCGAGAG  440005 ACGTTGGATGATGGCTCTAAAAAGCTGTCC ACGTTTGGATGCAGCCTTCTTCCTGATACAG  446614 ACGTTGGATGTAAAGCCCAGGGCTAAAGAC ACGTTGGATGAGAAGATGGCCAGAAAGGAG  451417 ACGTTGGATGCCAGAGTCATCGTTATCACC ACGTTGGATGCCTCACTAAGGATTCAACCC  454422 ACGTTGGATGCAGCTTTTGAGGCACTTTCC ACGTTGGATGAGCACCTTGCATACCCATAG  500277 ACGTTGGATGAGTTTCCCTACGTCTCTCTC ACGTTGGATGAGTAACTCTAGCCTCTGCTC  540717 ACGTTGGATGCATCCAAAACCCAACAAATCC ACGTTGGATGAGAGAGGTGTGTGACTTTTC  546106 ACGTTGGATGTTATAGCACTGATGGGCTCC ACGTTGGATGCTGTGACATACTTTTCCAGG  571039 ACGTTGGATGATTCCTGTAGCAGGCAACTG ACGTTGGATGGCTAGCTCTACTCTCTTCTC 1039542 ACGTTGGATGTGAGGTTCTGTCTGAACACC ACGTTGGATGTGGCTGCAATGGCTAACTTC 1039543 ACGTTGGATGATCTGACTCAGAAGAAGAGC ACGTTTGGATGGGCATTAATGGAGGTTATGC 1343180 ACGTTGGATGAGATGGCAACAGCAACACAG ACGTTGGATGCCAACAGCAGCTTCACAATC CHGB_SNP2 ACGTTGGATGAAATGGTATGTTTGTGTTTCC ACGTTGGATGTAATTTTTCCCCCCCAAATC rs384578 ACGTTGGATGAAATGGTATGTTTGTGTTCC ACGTTGGATGTAATTTTTCCCCCCCAAATC rs742710 ACGTTGGATGAGAAAGTGAGGAAGAGAGGG ACGTTGGATGATGAAATAGGCACGTGGCTC rs742711 ACGTTGGATGATGAAATAGGCACGTGGCTC ACGTTGGATGAGAAAGTGAGGAAGAGAGGG rs881118 ACGTTGGATGTATAGCTGAAGCCTGCTTTC ACGTTGGATGCAGTGAAGAGAAACACCTTG rs910122 ACGTTGGATGAAGGTGTTTCTCTTCACTGC ACGTTGGATGGGAGGGAGAGAACTATCAAA CHGB_SNP1 ACGTTGGATGTCACTCTGAGGTCTTGGAGC ACGTTGGATGTAAAGGGTTATCCAGGCGTC  180477 ACGTTGGATGGGAAGTAATTCTCTGGGCTG ACGTTGGATGAAGTGATCCTCCCACCTCAG  236102 ACGTTGGATGCAGCCTGTTCTCTCTGAAAC ACGTTGGATGGGATGCAAGAGGTTGTAGAG  236103 ACGTTGGATGCCTGTTTAAATCGTGGCTCC ACGTTGGATGAAACATAAGGAAGCTGAGGC  236106 ACGTTGGATGCAAGCCTTTGCAGCTCTATC ACGTTGGATGCCTCATAAGGGCCTTTGTAC  236107 ACGTTGGATGGAAGTTTACGTAAACTCTAG ACGTTGGATGGTGTGTGGCTTATTGTAGAG  236108 ACGTTGGATGGTATTTACTGTTGAACCCAG ACGTTGGATGATGTGGGTAAGTTGTGCACC  236109 ACGTTGGATGAGATTACAGGCACTAGCCAC ACGTTGGATGTCTGGGCAACATGGTGAAAC  236110 ACGTTGGATGATCGATCCAATGTTGACTGC ACGTTGGATGTTTTCAGAACAAACCCCACAG  236111 ACGTTGGATGTTCAGGAAGCAGCAACCATC ACGTTGGATGTATGCTGTGACCTCTCCAAC  236112 ACGTTGGATGAACGAGGTCAGGAGATCAAG ACGTTGGATGCACGCCCGGCTAATTTTTTC  236114 ACGTTGGATGGAACCAAGGAAGTCTGACTC ACGTTTGGATGAAAGCTACCAGTCATGTGCC  236115 ACGTTGGATGATCAAAGTCCATACTGCAGG ACGTTGGATGTATGATCGTAGGCACTGGAG  236116 ACGTTGGATGTGTTGTATTACCTGACCCTG ACGTTGGATGAAGCAAACCACTGAGTGTCC  236117 ACGTTGGATGCAATGGTGTGATCTTGCCTC ACGTTGGATGATTAGCCAAGTGTGGCAGTG  236118 ACGTTGGATGGGTTGAGTATCCCTAATCTG ACGTTGGATGCTTTCAGTGTCGTGTCAGGG  236121 ACGTTGGATGCAAGCTATGTCACAGTTTAAG ACGTTGGATGAGTCTTTTGCCCTTAATGTGG  236123 ACGTTGGATGATAATAAATTTAGACTTCAC ACGTTGGATGAAAATACTGGTGCGGCCAGA  236124 ACGTTGGATGGAATTTTGTTTGGCTCACGG ACGTTGGATGATTGCTGCTGGAAGCTTACC  236125 ACGTTGGATGCCATGCCTGAGTTATTTGC ACGTTGGATGATGGAGAAAGTAGATAGTAG  236148 ACGTTGGATGTAAGCCCAAGTGCTGTTGAG ACGTTGGATGCTCAGAAGTCTGATGTGTATC  236151 ACGTTGGATGTTGGCCTTTAGACTCCTGGG ACGTTGGATGAGAAGACACATAGCCGAGAG  236152 ACGTTGGATGAATAAAGGGTTATCCAGGCG ACGTTTGGATGTGGAGCCCTGTATTCTTCAC  236154 ACGTTGGATGTTCTGACAAGTTCCTGGCTG ACGTTGGATGGCTGCATTAGTCAACCTACC  236155 ACGTTGGATGGGTAGGTTGACTAATGCAGC ACGTTGGATGTGAGGTCCCGAACCAATTTC  236158 ACGTTGGATGAAACTCCTGACCTCGTGATC ACGTTGGATGCTCCTTAAGAAGATAGAGGC  236159 ACGTTGGATGGTCTCAAACTCCTGACCTCG ACGTTGGATGAAGAAGATAGAGGCAGCTGG  236161 ACGTTGGATGGATGTTGCCTCTAGGCTAGT ACGTTGGATGCACCATCTGACCTGTGCTAC  236165 ACGTTGGATGAAAATTAGCCATGCGTGGTG ACGTTGGATGTTCAAGCGGTTCTCCTGCCT  236168 ACGTTGGATGTCTATGTCTCCACTTGCATG ACGTTGGATGACACATTTTGCACACACACAC  236169 ACGTTGGATGGTGACTAGAATTTTTGTGTAC ACGTTGGATGGTGTGTGCAAATGTGTATCC  236171 ACGTTGGATGAACCTCCCACTTTGGCTTTC ACGTTGGATGGGTCCATTTAAAGCCTGGTG  236173 ACGTTGGATGATCAACCTGCACCACCAATC ACGTTGGATGGCTAAGATGGAAGTTGAAGTG  236175 ACGTTGGATGTTCTCCATCACTGCATCAAG ACGTTGGATGGTTATAGCCTGTATCGCAGC  236179 ACGTTGGATGCTAAATAACAGGTTTGACTC ACGTTGGATGGAACATTGAGAGTATCTTTAT  236181 ACGTTGGATGGTGAACATGTCTTTTCTGTAC ACGTTGGATGGGTAGAACCACTGTTTTTCG  236185 ACGTTGGATGGGGTCACTTGAATTCAGGAG ACGTTGGATGACTGCAACCACTGCCTCTTG  236187 ACGTTGGATGTAGTGAAACTCTGTCTCTGC ACGTTGGATGACCTGCACCAACCTTTAACC  236189 ACGTTGGATGTGGATTTACAGAAAAACTGC ACGTTGGATGCTGTGAGACACTAGGGATAC  364652 ACGTTGGATGTTTCTGCTGGGCTGTGATAG ACGTTGGATGGGGAAATGCTCAGCATGTAC  394604 ACGTTGGATGTATTTTGGGATGGTGTGGGC ACGTTGGATGGAACCAGGTCTTCCTTGATG  403727 ACGTTGGATGTCACTTGAACCCAGGAGATG ACGTTGGATGGTTTTGAGACAGAGTTTCGC  446658 ACGTTGGATGTCACTGAGTTCAACTCCTTC ACGTTGGATGGTTCCTGCTTTACCACTTCG  451571 ACGTTGGATGTTCTGGGTGGTTGCTCTCTG ACGTTGGATGAAGTAATGGCACACTGGAGG  452749 ACGTTGGATGTCCTACTCCAGTATGACCTC ACGTTGGATGGAAGTCCCAACCCCTAATAC  454328 ACGTTGGATGTGCAAACTGGTGCATCAGAG ACGTTGGATGCCTGGTATTTTCATATCGCC  742710 ACGTTGGATGGGCACATGGATATGGTGAAG ACGTTGGATGTGCCTCTGTGATGGTGTCCC  742711 ACGTTTGGATGGGCACATGGATATGGTGAAG ACGTTGGATGAAATAGGCACGTGGCTCCCC  881118 ACGTTGGATGTATAGCTGAAGCCTGCTTTC ACGTTGGATGTAGCAGTGAAGAGAAACACC  910122 ACGTTGGATGGTTTCTCTTCACTGCTATCT ACGTTGGATGACACGCCATTCTGAGAAGAG 1005517 ACGTTGGATGTACTAATGTCAGTGGTAGAG ACGTTGGATGTGAAGACACTGGCTGAAAAC 1394095 ACGTTGGATGTTCAGTGATCCAACTTCCGC ACGTTGGATGCCAAACTCCTTGATTGGC 2206817 ACGTTGGATGGCAGAAACCCAGTGAAGTAG ACGTTGGATGAAACCAGTTACTAACTGTAG 2268339 ACGTTGGATGATCCTGGAGATGTTATACCC ACGTTGGATGCCTGGTGTTTAAGGCTCAAC 2300427 ACGTTTGGATGAGATTACAGGCATGAGCCAC ACGTTGGATGAAGTTAAATAAGCTCTTCTG 2326680 ACGTTGGATGAGGCTAATTCCTTCTCCTGG ACGTTGGATGTCGTGCAACATCACTGTGTC 2423131 ACGTTGGATGATGCCTGCCTTACGAGAATG ACGTTGGATGTGTCACTAGAATATGTGAAC 2876003 ACGTTGGATGGCAAAGACTAAGAGTCTGTAG ACGTTGGATGCTGAGCCAGATTCTGACATT 3761873 ACGTTGGATGCTGTCCCTCTTAGAGCAATG ACGTTGGATGCTATGAGCCTTTGACACAGC

TABLE 20 dbSNP Extend Term rs# Primer Mix   54144 CTGAAAGACACCATTTAT CGT  183535 GTCCTGCACAAGACTTTGATA ACG  236104 CCCATTTCATACCACCTATCA ACG  236105 CTCCCTCCTCCTTGAGACC ACT  236113 GATCATTCATGAAACAGATTCTA ACG  236119 TGTTCTCAAGGAAAAAAGAAAAAA ACT  236120 GATCATCCCTGGGAATGGTA ACT  236122 GAGGCAGGGAATCAGCAATA ACT  236143 ACCCCAAATAGGTAAAGATCTGT ACT  236145 CTCCTGCACTGAGCTCCTAT ACT  236146 TATCTCCGCCCTAAGAATACT ACT  236149 GAAATATTAGAATTTAGAGGCAG ACT  236160 GAGTTTTTATGAGAAAGGGCAA ACT  236162 GTTGTTTTAAAGTGTTGGTTGTAA ACT  236163 CAATACATAGTGAAGCTTTGGG ACT  236164 CTGGTGTCACACACACATGTA ACT  236166 GGAACATCTCAGAAAAAAAA CGT  236167 CTTGTGCAGGACTTAGACCA ACT  236176 ATAGGCTTTCTTGTGTATTTGCA ACT  236177 AGTGAGGGGAAGCAGAGTC CGT  236180 ACTTGGGAATTAGGTGGAGG ACG  236182 GTTCAGAGATAATGCTGCTGATC CGT  236183 GAAATCCCACAGGAACACAAT ACT  236184 CCCAGCAAATAACAAGAATTGGCC ACT  379418 CTTAAGCCAAGACAAACA ACG  400735 TTCATCTTCCACCCTGGCC ACT  409035 TGCTTTTGCTTGCCTCCCACA ACG  440005 GCTGTCCTTITTACAAGGAAAT ACT  446614 TAAAGACTGAAGCTTTCACAGT ACT  451417 CGTTATCACCATTGGGCTTTA CGT  454422 GATCCTTCTCACTTACTGTTC ACT  500277 GATTATGCCCTGAGGTCTTTTG ACT  540717 AACCCAACAAATCCTAGGGC ACG  546106 GATGGGCTCCCCATATGAC ACT  571039 TGTAGCAGGCAACTGAGCAGGAGA CGT 1039542 GAACACCCTCCAGCACAAG ACT 1039543 AGAAGAGCTTTCATCTGTGTG ACT 1343180 CACAGCCCTCCATTACAGC ACT CHGB_SNP2 GTATGTTTGTGTTCCATTTGCA ACT rs384578 GTATGTTTGTGTTCCATTTGCA ACT rs742710 GAAGAGAGGGGCCTTGAGC ACG rs742711 TCCCCTGCCTCTGTGATGG ACG rs881118 CTGAAGCCTGCTTTCTTTCAT ACT rs910122 CTTCACTGCTATCTTCCCCT ACG CHGB_SNP1 TCTTGGAGCCCTGTATTC ACG  180477 GTGGCTCACGCCTATAA CGT  236102 GTTCTCTCTGAAACCTGTTA ACT  236103 CATGCACCAGCTGTGTG ACT  236106 GAACATTCCAGGCAAAC ACT  236107 GTTCTGGTAAAAAAAAAGTTTG CGT  236108 CTGTTGAACCCAGAAATATC ACT  236109 CACTAGCCACCACGCCC ACT  236110 CAATGTTGACTGCATTGACT ACT  236111 GTTTCTGAGGTTACCAGA ACT  236112 ACCATCCTAGCTAACACG ACT  236114 AATCACAAGTACCTCGAATAC ACT  236115 AGGTAAGTGGCAGAACT ACT  236116 TCAGGCAAGCACAGTACAAA ACG  236117 GCCTCCCAAGTAGCTGG ACT  236118 CCCTAATCTGAAAATCTGAAATCT ACT  236121 AAGAATTTTCTTATTCAACTGTC ACT  236123 CTTCACTAAATAAAAATGTGTCC ACG  236124 GTTTTGGCTCACGGAATTAT ACG  236125 TTTAACTCCTAGCTTTTAAAGA ACG  236148 AAATGTGGCTGGTCCGATCTG ACT  236151 ATTCTCCTGGCTCCCTG ACG  236152 TTATCCAGGCGTCCAGG ACT  236154 TGATGCCACTGGTCAGG ACT  236155 AATTCCCCTTTGCACTCAT ACT  236158 TTACAACTGTAAGCCACCGC ACT  236159 CCTGACCTCGTGATCTG ACG  236161 CTCTAGGCTAGTATTAATTTTTGT ACT  236165 TAACGCCTGTAATCCCA ACT  236168 ACTTGCATGTGTATGTATATATCT ACT  236169 ATGTCTTTTCCCCCTCT ACT  236171 AAGTGCTGGGATTACAGATA ACG  236173 TTGCTCCCTCTCCCCTT ACT  236175 CACTGCATCAAGATGGGCC ACG  236179 CAGGTTGACTCAAAACTTTAA ACG  236181 ATGTCTTTTCTGTACTGGATA ACT  236185 TACTGAGGAGGCTGAGG ACG  236187 AACTCTGTCTCTGCAAAAAAA ACT  236189 CAGAAAAACTGCACAAAAA ACT  364652 CTGTGATAGGAAAAAAGGAA ACT  394604 CCAGCAGAGGCAAAAATAAGA ACG  403727 TGCCACTGCACTCCAGCCT ACT  446658 AGGAAAAGAGAGGCAAAC ACT  451571 GCTGTCTTCATTCTCTTTGT ACG  452749 CCTATTTTCAAGTCAGGT ACG  454328 CCTAAACAGCAGTTTTAGTACAT ACG  742710 AGAGAGGGGCCTTGAGC ACG  742711 TGAGCCGGGAAAGGGAC ACT  881118 TGAAGCCTGCTTTCTTTCAT ACT  910122 CTTCACTGCTATCTTCCCCT ACG 1005517 GGTAGAGAATGTAATAACAGT ACT 1394095 ACGAGAGGGGCGGGGCG ACT 2206817 TTAGAGCAGGGCAGGGG ACT 2268339 CAGAATGCTGAGATGGC ACT 2300427 CACCCGGCCGGGAAAAT ACT 2326680 TGGAATTTGAGAAGGCCTG ACT 2423131 GCCTTACGAGAATGTTTATTT CGT 2876003 AGAGTCTGTAGTCCCAA ACT 3761873 TGTATTTTCCATAGTAATTTGCTC ACT Genetic Analysis and Allelotyping Results

Allelotyping results are shown for cases and controls in Table 21. The allele frequency for the A2 allele is noted in the fifth and sixth columns for breast cancer pools and control pools, respectively, where “AF” is allele frequency. The allele frequency for the Al allele can be easily calculated by subtracting the A2 allele frequency from 1 (A1 AF=1−A2 AF). For example, SNP rs2300427 has the following case and control allele frequencies: case A1 (T)=0.615; case A2 (G)=0.385; control A1 (T)=0.605; and control A2 (G)=0.395, where the nucleotide is provided in paranthesis. SNPs with blank allele frequencies were untyped (“not AT”). TABLE 21 dbSNP Position Chrom Alleles A2 Control rs# in FIG. 3 Position (A1/A2) A2 Case AF AF p-Value 2300427  186 5842586 T/G 0.395 0.385 0.7201 571039 1332 5843732 G/T 0.669 0.695 0.3506 236143 1893 5844293 T/C 0.499 0.545 0.1289 236145 2786 5845186 C/G 0.198 0.188 0.6666 236146 2962 5845362 A/G 0.549 0.601 0.0802 446658 3377 5845777 A/C 0.013 0.011 0.8118 500277 5522 5847922 A/G 0.217 0.214 0.8990 2268339  5621 5848021 A/G 0.931 0.888 0.0128 454328 5889 5848289 G/A 0.330 0.323 0.7912 236148 7531 5849931 T/C 0.463 0.459 0.9081 236149 8268 5850668 T/C 0.550 0.553 0.9289 910122 8923 5851323 G/A 0.373 0.337 0.2228 881118 8988 5851388 A/C 0.026 0.042 0.1278 236151 9117 5851517 G/A 0.295 0.272 0.3986 236152 9448 5851848 C/G 0.310 0.316 0.8329 CHGB_SNP1 9494 5851894 G/A 0.497 0.485 0.6989 742710 9628 5852028 G/A 0.010 0.044 0.0007 742711 9640 5852040 T/C 0.326 0.266 0.0282 236154 11072 5853472 T/G 0.410 0.421 0.6955 236155 11150 5853550 A/G 0.454 0.445 0.7568  54144 11379 5853779 C/A 0.326 0.350 0.3946 540717 11692 5854092 G/A 0.492 0.495 0.9036 236158 12056 5854456 T/C 0.251 0.229 0.3815 236159 12104 5854504 G/A 0.404 0.385 0.5205 446614 14160 5856560 T/C 0.968 0.951 0.1381 1343180  14836 5857236 A/G 0.376 0.383 0.7937 1039542  14980 5857380 A/C 0.348 0.390 0.1491 400735 15165 5857565 A/G 0.286 0.272 0.5984 1039543  15315 5857715 A/G 0.356 0.318 0.1914 440005 15624 5858024 A/C 0.563 0.575 0.6852 394604 15796 5858196 C/T 0.297 0.277 0.4715 546106 15939 5858339 T/C 0.733 0.714 0.4816 452749 16581 5858981 C/T 0.297 0.262 0.1967 364652 17045 5859445 T/C 0.513 0.558 0.1426 403727 18501 5860901 A/G 0.743 0.748 0.8547 236160 21800 5864200 A/G 0.893 0.794 0.0000 236161 21966 5864366 A/G 0.121 0.187 0.0026 236162 22134 5864534 A/G 0.735 0.648 0.0021 236163 22181 5864581 A/G 0.042 0.096 0.0006 236164 23028 5865428 A/G 0.815 0.734 0.0018 236165 23312 5865712 A/G 0.888 0.892 0.8319 236166 23573 5865973 T/A 0.060 0.125 0.0004 236167 23858 5866258 A/G 0.695 0.675 0.4700 183535 23888 5866288 G/A 0.088 0.149 0.0020 236168 23990 5866390 T/C 0.302 0.303 0.9685 236169 24073 5866473 A/G 0.048 0.115 0.0001 236171 25330 5867730 G/A 0.749 0.739 0.6961 236173 26473 5868873 T/C 0.051 0.119 0.0002 236175 27958 5870358 C/T 0.172 0.289 0.0000 236176 28421 5870821 A/G 0.056 0.143 0.0000 451571 28804 5871204 C/T 0.954 0.924 0.0378 236177 29322 5871722 C/A 0.072 0.125 0.0040 1005517  30819 5873219 G/C 0.970 0.934 0.0051 236179 31956 5874356 G/A 0.056 0.108 0.0021 236180 32592 5874992 G/A 0.073 0.124 0.0047 236181 32818 5875218 G/C 0.908 0.807 0.0000 236182 32880 5875280 G/T 0.914 0.844 0.0006 236183 33244 5875644 G/C 0.870 0.771 0.0001 236184 33845 5876245 A/G 0.059 0.120 0.0006 236185 34272 5876672 G/A 0.028 0.041 0.2547 236187 34931 5877331 T/C 0.048 0.088 0.0090 1394095  36870 5879270 T/G 0.880 0.884 0.8136 236189 37790 5880190 T/C 0.105 0.160 0.0077 236110 38708 5881108 T/G 0.875 0.799 0.0009 236111 39135 5881535 T/C 0.045 0.080 0.0161 236112 39919 5882319 A/G 0.852 0.854 0.9265 236113 40166 5882566 C/T 0.869 0.790 0.0007 236114 40985 5883385 A/G 0.345 0.347 0.9357 236115 41049 5883449 A/G 0.835 0.749 0.0007 236116 41935 5884335 C/T 0.679 0.677 0.9439 236117 42775 5885175 A/C 0.926 0.903 0.1611 236118 43807 5886207 T/C 0.771 0.697 0.0060 236119 44254 5886654 A/G 0.773 0.694 0.0032 3761873  44814 5887214 A/C 0.044 0.042 0.8940 236120 45249 5887649 T/G 0.072 0.109 0.0291 451417 47599 5889999 C/A 0.086 0.103 0.3196 379418 47807 5890207 G/A 0.102 0.166 0.0022 2326680  48555 5890955 A/C 0.610 0.542 0.0233 409035 49249 5891649 G/A 0.788 0.700 0.0012 454422 49293 5891693 A/C 0.763 0.673 0.0013 236102 57566 5899966 A/C 0.856 0.780 0.0014 236103 63587 5905987 T/C 0.798 0.740 0.0205 236104 64560 5906960 C/T 0.793 0.714 0.0029 236105 65432 5907832 C/G 0.077 0.127 0.0065 236106 66291 5908691 T/C 0.109 0.154 0.0265 236107 71331 5913731 A/T 0.063 0.103 0.0159 180477 73344 5915744 A/T 0.768 0.698 0.0086 236108 74159 5916559 C/T 0.825 0.742 0.0012 236109 74564 5916964 T/C 0.162 0.170 0.7214 236121 78194 5920594 A/G 0.136 0.182 0.0345 236122 79128 5921528 T/C 0.862 0.790 0.0019 236123 79393 5921793 C/T 0.742 0.725 0.5078 236124 81579 5923979 G/A 0.867 0.800 0.0029 236125 82574 5924974 C/T 0.069 0.104 0.0385 2876003  85309 5927709 G/C 0.847 0.836 0.6067 CHGB_SNP2 87076 5929476 A/G 0.783 0.734 0.0538 2423131  87844 5930244 C/A 0.471 0.427 0.1473 2206817  90241 5932641 T/C 0.974 0.985 0.2027

FIG. 16 shows the proximal SNPs in and around the CHGB and C20orf154 gene region. As indicated, some of the SNPs were untyped. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group. The minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in FIG. 16 can be determined by consulting Table 18. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.

To aid the interpretation, multiple lines have been added to the graph. The broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01. The vertical broken lines are drawn every 20 kb to assist in the interpretation of distances between SNPs. Two other lines are drawn to expose linear trends in the association of SNPs to the disease. The light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W. S. Cleveland, E. Grosse and W. M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J. M. Chambers and T. J. Hastie, Wadsworth & Brooks/Cole.). The black line (or generally top=most curve, e.g., see peak in left-most graph just to the left of position 92150000) provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10 kb sliding window with 1 kb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10⁻⁸ were truncated at that value.

Finally, the gene or genes present in the loci region of the proximal SNPs as annotated by Locus Link (http address: www.ncbi.nim.nih.gov/LocusLink/) are provided on the graph. The exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3′ end of each gene to show the direction of transcription.

Additional Genotyping

In addition to the CHGB/C20orf154 incident SNP, two other SNPs (rs742710 and rs236110) were genotyped in the discovery cohort and found to be significantly associated with breast cancer. See Table 24.

The methods used to verify and genotype the proximal SNP of Table 21 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 22 and Table 23, respectively. TABLE 22 dbSNP First Second rs# PCR primer PCR primer 742710 ACGTTGGATGGACATGAAATAGGCACGTGG ACGTTGGATGAGAAAGTGAGGAAGAGAGGG 236110 ACGTTGGATGTCACTCTGTTTCTACTAACC ACGTTGGATGATCGATCCAATGTTGACTGC

TABLE 23 dbSNP Extend Term rs# Primer Mix 742710 AGAGAGGGGCCTTGAGC ACG 236110 AATGTTGACTGCATTGACT ACT

Table 24, below, shows the case and control allele frequencies along with the p-values for the SNPs genotyped. The disease associated allele of column 4 is in bold and the disease associated amino acid of column 5 is also in bold. For rs742710 the proline is associated with breast cancer, and for rs236110, the glutamine is associated with breast cancer. The chromosome position provided corresponds to NCBI's Build 33. TABLE 24 Genotpying Results Amino dbSNP Position in Chromosome Alleles Acid AF AF Odds rs# Position (A1/A2) Change F case F control p-value Ratio 742710 9628 5852028 G/A P413L A = 0.030 A = 0.060 0.0279 0.51 G = 0.970 G = 0.940 236110 38708 5881108 T/G Q63K T = 0.080 T = 0.130 0.0112 1.66 G = 0.920 G = 0.870

Example 6 LOC338749 Proximal SNPs

It has been discovered that a polymorphic variation (rs763471) in the LOC338749 gene region is associated with the occurrence of breast cancer (see Examples 1 and 2). Subsequently, SNPs proximal to the incident SNP (rs763471) were identified and allelotyped in breast cancer sample sets and control sample sets as described in Examples 1 and 2. Approximately sixty-three allelic variants located within the LOC338749 region were identified and allelotyped. The polymorphic variants are set forth in Table 25. The chromosome position provided in column four of Table 25 is based on Genome “Build 33” of NCBI's GenBank. TABLE 25 dbSNP Position in Chromosome Allele rs# Chromosome Position Variants 2957666 11 142 10442142 A/G 2198010 11 693 10442693 A/C 2198009 11 731 10442731 T/C 2198008 11 879 10442879 T/G 2957667 11 1084 10443084 C/A 2957669 11 2249 10444249 C/G 2957670 11 2519 10444519 A/G 2923115 11 4461 10446461 G/A 2957679 11 4616 10446616 A/G 2957678 11 5109 10447109 G/A 2957677 11 5270 10447270 C/G 2923117 11 5436 10447436 G/C 2957675 11 5457 10447457 T/C 750371 11 6536 10448536 G/C 1562781 11 9665 10451665 T/A 752373 11 16120 10458120 C/T 3741045 11 29489 10471489 T/C 3741044 11 29524 10471524 G/T 763470 11 49159 10491159 A/G 763471 11 49273 10491273 G/T 1376001 11 49596 10491596 A/G 1376000 11 50135 10492135 A/G 1450274 11 50184 10492184 C/G 1375999 11 50393 10492393 A/C 1450273 11 50401 10492401 T/G 1450270 11 55750 10497750 T/C 899013 11 73843 10515843 T/C 2071019 11 73852 10515852 T/C LOC_SNP1 11 74052 10516052 G/A LOC_SNP2 11 75382 10517382 A/G 930672 11 75662 10517662 T/A 922359 11 75942 10517942 T/C 2403330 11 77917 10519917 A/G 936513 11 78821 10520821 A/G 3891547 11 94813 10536813 G/G 3741043 11 97149 10539149 T/C Assay for Verifying and Allelotyping SNPs

The methods used to verify and allelotype the proximal SNPs of Table 25 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays provided in Table 26 and Table 27, respectively. TABLE 26 dbSNP Forward Reverse rs# PCR primer PCR primer 752373 ACGTTGGATGTATCACAAGAACAGCATGGG ACGTTGGATGATGGTTTCTGTAATCCCCCC 763470 ACGTTGGATGAAGAGGAGTGGCTGATAATG ACGTTTGGATGAAGCAGAAAACTTTGTGCCG 763471 ACGTTGGATGGGCAGGTCATGGATTTATTG ACGTTTGGATGCATCATTCCTCTGTGAGGCG 763471 ACGTTGGATGGTGAAGAGCTCTGAAATGCC ACGTTGGATGTAACTCCTGTGTGGCTTTCT 899011 ACGTTGGATGAAGGTGGAGCCTGCCTCAAG ACGTTGGATGAGCTTTGCACCCTGTGATGC 899011 ACGTTGGATGAAGGTGGAGCCTGCCTCAAG ACGTTGGATGAGCTTTGCACCCTGTGATGC 922359 ACGTTGGATGTCAAGCGATCCTCTTCAGCC ACGTTGGATGATTCATTCCAAGACCGGGTG 930672 ACGTTGGATGGTGGGTTACTTGGTCCATAC ACGTTGGATGACAGAGCAAGACCTTCTCTC 936513 ACGTTGGATGGTATGAAGTTCTTGCAGAGT ACGTTGGATGTACTACTGCACTCCAGCCTG 1375999 ACGTTGGATGCCATTCTTTTACCTTGAACC ACGTTGGATGCAGAGACTTGCAGAATGGAC 1376000 ACGTTGGATGATAGCTGATGGTGTGCTGAG ACGTTGGATGAAGCTTGCCTCCCAAGTTAG 1376001 ACGTTGGATGCAAACAATCCCATTACACAG ACGTTGGATGCAGTACAACAGGGTGGCTATC 1450270 ACGTTGGATGCCATATCACATGGATATGAGG ACGTTGGATGCATGGCTTCTCTTACACCTG 1450273 ACGTTGGATGGCTGCATATAAGAGACACATG ACGTTGGATGGCCACTCCAGCTTTCTTTTG 1450274 ACGTTGGATGTGAGAGGAAGCCTGGTGTTG ACGTTGGATGAAGCTTGCCTCCCAAGTTAG 1562781 ACGTTGGATGTATGTCTCCTGCCTTCTTCC ACGTTGGATGGGAAAGAAGCTTGATGTGGC 2071019 ACGTTGGATGGGTAAACAACTGACCCATCC ACGTTGGATGCCTGGGAAATAACCATGAGC 2071020 ACGTTGGATGAATTCACAGCTAAGCCTCCC ACGTTGGATGTTCAGCTCCAGCTGCATGTT 2198008 ACGTTGGATGGTAGAAGTTTAGTATATGATG ACGTTGGATGCCCTGTCATTTCAAATACCG 2198009 ACGTTGGATGCTTGTGCCAATCCCACAATG ACGTTGGATGGCAGAAGTCTAGCCAAGAAC 2198010 ACGTTGGATGCTTGTGCCAATCCCACAATG ACGTTGGATGAATGCAGAAGTCTAGCCAAG 2403330 ACGTTGGATGTAACTCTGAGACCCAAGGAC ACGTTGGATGCCAGACAGTTGTGTGTTGAC 2923115 ACGTTGGATGGGATTACCCTAAGGATCCAC ACGTTGGATGAGAGGAATTCAGTTGCTGCC 2923117 ACGTTGGATGTTGAGTCCAAGAGGTTGAGG ACGTTGGATGAGACAGTCTTGCTCTGTCAC 2957666 ACGTTGGATGTACTTGGGAGACTGAGGTAG ACGTTGGATGGCATAGTGGTGTGATCATGG 2957667 ACGTTGGATGAGAATGGTCTTTCCCACTCC ACGTTGGATGATGGATTACGGAAGGAATAC 2957669 ACGTTGGATGTACTGAGACTCCCAGCATTG ACGTTGGATGGTGTGCAGCTTAGTAAGTGC 2957670 ACGTTGGATGTCATGTGATTCTCCTGCCTC ACGTTGGATGGTGAAACCCCGTCTCTACTA 2957675 ACGTTGGATGAGAATGACTTGGGTTTTGGG ACGTTGGATGCAGTGAGTTGTGACAGCACC 2957677 ACGTTGGATGGTCTTTCTCAATCCCAGCAC ACGTTGGATGACGAGATCTCCTTGTGTTGC 2957678 ACGTTGGATGAAGACCTCAGGATGTGATGC ACGTTGGATGATGACCCCGTTTCTTTGCAC 2957679 ACGTTGGATGAGTTCGTCAGAGAGATGTCC ACGTTGGATGGAGCACATGGATTCACAGAG 3741043 ACGTTGGATGGACATCAGAAGCTAATTGGG ACGTTGGATGCTTCTTAATGGTAGGGCCAG 3741044 ACGTTGGATGTTTGTTATGCAGAGGTGGCC ACGTTGGATGTAGATGGGCTCTTCTTGGAC 3741045 ACGTTGGATGAACTGAGCTTCAGACTTCCC ACGTTGGATGTCAGACCTGTAGATGGGCTC 3891547 ACGTTGGATGGCCATCAAGTTTGTGGCAAT ACGTTGGATGAAGCTATATGGAGCCCAAGG

TABLE 27 dbSNP Extend Term rs# Primer Mix 752373 GGGACCAGGTGGAGATAA ACG 763470 AGAAAACTTTGTGCCGTTTTCT ACT 763471 CCAGGCAGCAACTCCCT ACT 763471 CTCCAAGCAGTAAAGATGTTC CGT 899011 TTGGTTTTAGAGGATTGCTCC ACG 899011 GGTTTTAGAGGATTGCTCC ACG 922359 TCATGCCTATAATCCAAGCA ACT 930672 AAAAGCAAGAAACAACAGCA CGT 936513 AGACAGGGTGAGACCTC ACT 1375999 TATGCTGCATATAAGAGACACAT ACT 1376000 ACATATTTCTGGTCTCCA ACT 1376001 ACAGGGTGGCTATCATTAAC ACT 1450270 TGTGAACTGAAAAGTCAAG ACT 1450273 CTTGAACCTATTTCTGTTTTT ACT 1450274 CTCCCAAGTTAGATTGGTTTA ACT 1562781 CTTGATGTGGCTGAAGT CGT 2071019 GTGCTGTTGAAATCCTGGG ACT 2071020 AACCCTTTGTCAGCTGAA ACT 2198008 CAGGCTTTTGGCTAAGATCAAG ACT 2198009 AGTGAAGAATTTTCCCTATTAGAT ACT 2198010 AAGTCTAGCCAAGAACATTT ACT 2403330 CTCCCACTCCTCTCATCAG ACT 2923115 GTTGCTGCCCGCTTTCC ACG 2923117 CTCTGTCACCCATGCTGGA ACT 2957666 ACCTCCTGGGCTCAAGC ACT 2957667 GGAAGGAATACTAAAGAACAA CGT 2957669 AGTAAGTGCTGTGATGCACC ACT 2957670 TAGCTGAGCATGGTGGC ACT 2957675 AGCATGGGTGACAGAGC ACT 2957677 TGTGTTGCCCAGACTAG ACT 2957678 ACTCCCTGGCCTCCCCT ACG 2957679 TCACAGAGCTGCCAGGG ACT 3741043 CAAAATTCTCCTGCCAC ACT 3741044 GGGGAAAGGGAAGTCTG CGT 3741045 TAGATGGGCTCTTCTTG ACT 3891547 TATGGAGCCCAAGGATGACC ACT Genetic Analysis and Allelotyping Results

Allelotyping results are shown for cases and controls in Table 28. The allele frequency for the A2 allele is noted in the fifth and sixth columns for breast cancer pools and control pools, respectively, where “AF” is allele frequency. The allele frequency for the A1 allele can be easily calculated by subtracting the A2 allele frequency from 1 (A1 AF=1−A2 AF). For example, the SNP rs2957666 has the following case and control allele frequencies: case A1 (A)=0.863; case A2 (G)=0.137; control A1 (A)=0.855; and control A2 (G)=0.145, where the nucleotide is provided in paranthesis. SNPs with blank allele frequencies were untyped. TABLE 28 dbSNP Position in Chromosome A1/A2 A2 Control rs# Position Allele A2 Case AF AF p-Value 2957666 142 10442142 A/G 0.145 0.137 0.6913 2198010 693 10442693 A/C 0.424 0.410 0.6335 2198009 731 10442731 T/C 0.485 0.469 0.6093 2198008 879 10442879 T/G 0.424 0.422 0.9461 2957667 1084 10443084 C/A 0.437 0.430 0.8105 2957669 2249 10444249 C/G 0.556 0.552 0.8955 2957670 2519 10444519 A/G 0.051 0.054 0.8305 2923115 4461 10446461 G/A 0.490 0.475 0.6091 2957679 4616 10446616 A/G 0.391 0.403 0.6947 2957678 5109 10447109 G/A 0.458 0.447 0.7210 2957677 5270 10447270 C/G 0.479 0.475 0.9147 2923117 5436 10447436 G/C 0.913 0.950 0.0164 2957675 5457 10447457 T/C 0.425 0.421 0.8857  750371 6536 10448536 G/C 0.143 0.174 0.1547 1562781 9665 10451665 T/A 0.966 0.973 0.4889  752373 16120 10458120 C/T 0.251 0.195 0.0255 3741045 29489 10471489 T/C 0.715 0.719 0.8787 3741044 29524 10471524 G/T 0.419 0.374 0.1272  763470 49159 10491159 A/G 0.139 0.137 0.9405  763471 49273 10491273 G/T 0.481 0.537 0.0661 1376001 49596 10491596 A/G 0.538 0.484 0.0705 1376000 50135 10492135 A/G 0.228 0.256 0.2849 1450274 50184 10492184 C/G 0.777 0.736 0.1185 1375999 50393 10492393 A/C 0.933 0.913 0.2078 1450273 50401 10492401 T/G 0.845 0.817 0.2102 1450270 55750 10497750 T/C 0.349 0.264 0.0025  899013 73843 10515843 T/C 0.388 0.385 0.9145 2071019 73852 10515852 T/C 0.824 0.782 0.0756 LOC_SNP1 74052 10516052 G/A 0.245 0.217 0.2760 LOC_SNP2 75382 10517382 A/G 0.283 0.327 0.1112  930672 75662 10517662 T/A 0.157 0.154 0.8763  922359 75942 10517942 T/C 0.106 0.120 0.4749 2403330 77917 10519917 A/G 0.624 0.574 0.0959  936513 78821 10520821 A/G 0.381 0.423 0.1554 3891547 94813 10536813 C/G 0.069 0.079 0.5157 3741043 97149 10539149 T/C 0.855 0.832 0.2972

FIG. 17 shows the proximal SNPs in and around the LOC338749 region for females. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group. The minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in FIG. 17 can be determined by consulting Table 28. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.

To aid the interpretation, multiple lines have been added to the graph. The broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01. The vertical broken lines are drawn every 20 kb to assist in the interpretation of distances between SNPs. Two other lines are drawn to expose linear trends in the association of SNPs to the disease. The light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W. S. Cleveland, E. Grosse and W. M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J. M. Chambers and T. J. Hastie, Wadsworth & Brooks/Cole.). The black line (or generally top-most curve, e.g., see peak in left-most graph just to the left of position 92150000) provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10 kb sliding window with 1 kb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10⁻⁸ were truncated at that value.

Finally, the gene or genes present in the loci region of the proximal SNPs as annotated by Locus Link (http address: www.ncbi.nlm.nih.gov/LocusLink/) are provided on the graph. The exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3′ end of each gene to show the direction of transcription.

Example 7 TTN/LOC351327 Proximal SNPs

It has been discovered that a polymorphic variation (rs2046778) in the TTN/LOC351327 region is associated with the occurrence of breast cancer (see Examples 1 and 2). Subsequently, SNPs proximal to the incident SNP (rs2046778) were identified and allelotyped in breast cancer sample sets and control sample sets as described in Examples 1 and 2. Approximately forty-six allelic variants located within the TTN/LOC351327 region were identified and allelotyped. The polymorphic variants are set forth in Table 29. The chromosome position provided in column four of Table 29 is based on Genome “Build 33” of NCBI's GenBank. TABLE 29 dbSNP Position in Chromosome Allele rs# Chromosome Position Variants 2291309 2 200 179587600 T/G 2291310 2 381 179587781 T/C 1484119 2 5303 179592703 G/C TTN_SNP1 2 6084 179593484 C/T 1484120 2 6879 179594279 T/A 2291312 2 7837 179595237 T/C 3816782 2 7985 179595385 C/A 2291313 2 9333 179596733 T/C 2306636 2 11559 179598959 T/C 2291304 2 12473 179599873 T/C 2291305 2 12880 179600280 T/A 1905520 2 13606 179601006 C/T 2291306 2 14861 179602261 A/G TTN_SNP2 2 20658 179608058 C/T 2054708 2 22200 179609600 G/A 2306637 2 24525 179611925 A/C 3769863 2 26373 179613773 T/G 3769860 2 42869 179630269 A/T 3816849 2 43713 179631113 A/G 3769858 2 44429 179631829 A/G 2279472 2 49037 179636437 A/G 2046778 2 49170 179636570 A/G 1565288 2 50206 179637606 G/A 2129108 2 51552 179638952 C/T 2170850 2 51674 179639074 T/G 2029397 2 56427 179643827 T/C 2029395 2 56844 179644244 G/A 1844334 2 57953 179645353 A/G 998329 2 60862 179648262 G/A 1489486 2 61606 179649006 T/C 2046777 2 62560 179649960 G/A 1489483 2 65078 179652478 A/G 1489482 2 65155 179652555 G/T 2366911 2 70295 179657695 T/C 2366912 2 70335 179657735 G/T 2366913 2 70398 179657798 C/T 2078403 2 79233 179666633 C/T 1489481 2 80025 179667425 C/G 2129111 2 84521 179671921 A/G 966783 2 84540 179671940 C/T 1489480 2 85170 179672570 T/G 1489479 2 85300 179672700 A/C 726215 2 87596 179674996 A/C 1387472 2 89696 179677096 C/A 2086832 2 92219 179679619 A/T 1872203 2 96589 179683989 A/T Assay for Verifying and Allelotyping SNPs

The methods used to verify and allelotype the proximal SNPs of Table 29 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 30 and Table 31, respectively. The methods used to verify and allelotype the proximal SNPs of Table 29 are the same methods described in Examples 1 and 2 herein. The PCR primers and extend primers used in these assays are provided in Table 30 and Table 31, respectively. TABLE 30 dbSNP Forward Reverse rs# PCR primer PCR primer 726215 ACGTTGGATGAACTGAGCCCCATGAAATGC ACGTTGGATGAAAACAGCAATTGAGAACAC 966783 ACGTTGGATGCTCCTGAATTTTAGCCATAC ACGTTGGATGTACGCAATAGTTCCTGGGAG 998329 ACGTTGGATGGAAGAGCACATTATTGCTGG ACGTTGGATGACACACTGGTGTTTTGTCAG 1387472 ACGTTGGATGGAAAGGCCTTGAATTGGAAC ACGTTGGATGGTTCTGCTAGTGTCATCTTC 1484119 ACGTTGGATGGCAGCTACAATCATAAAGGG ACGTTGGATGTGTGCCCTTAATAATGGTTG 1484120 ACGTTGGATGATGGTCATGGCATCCAGTTC ACGTTGGATGGGCTGGTTTCTGACACTATC 1489479 ACGTTGGATGGGAGCATCAGTCATTTTGGG ACGTTGGATGCACCAGGACATAACATGACG 1489480 ACGTTGGATGGGGTTGTGGAGAATCATTAC ACGTTGGATGGGTGGCAGTAATCTTCACTT 1489481 ACGTTGGATGTCTCTGCAGTTGAGGAGATG ACGTTGGATGTTGGGAAAGGCCATCAAGTC 1489482 ACGTTGGATGCTCTGGATAAAAGACTCAGC ACGTTGGATGCCCTTCCAACAGCTATCTGG 1489483 ACGTTGGATGTTGGTTTGCTATCAATGAAG ACGTTGGATGGATAGGTGTACACATATAGC 1489486 ACGTTGGATGAAAAAACACACCACAGCCCC ACGTTGGATGCTTCGTATTFGGCTCTGACC 1552280 ACGTTGGATGATGAAAAGTGACACCCATCC ACGTTGGATGTCTGAAGCTGTTGAATCAGG 1565288 ACGTTGGATGTAGCCAATTGGTGAACACTC ACGTTGGATGCTGCCAGTCATAAGGCAAAG 1844334 ACGTTGGATGGCCAAGGAAACTAATTCCTG ACGTTGGATGCACTTTTGGAAGACAGTTCGG 1872203 ACGTTGGATGGTTGCATTAGCTGTTATTCTC ACGTTGGATGCCAGCAATTCTATTTCAGAG 1905520 ACGTTGGATGCATGGTTTATACTTACTTACG ACGTTGGATGGTTTATTCCTGTTTCCACAC 2029395 ACGTTGGATGGGAGGGAGACAAAGATTCAC ACGTTGGATGGCAACAGTTTCACCTTTGGC 2029397 ACGTTGGATGCTCACAGTCCTGAAGACTTG ACGTTGGATGTGGAAGTGAAGGAGAGAAGC 2046777 ACGTTGGATGGGACTTCAAATATGGTTCAC ACGTTGGATGTTAAGCCTGGGACTTTTGGG 2046778 ACGTTGGATGGTTCCCTTCCCCCATAAAAC ACGTTGGATGCATGAAGCCTTATGCTTGAG 2054708 ACGTTGGATGCTAGGCATATCATGCCTCTG ACGTTGGATGTTGAGCTCACTGTTACCTGC 2078403 ACGTTGGATGTGTGCTCAGGATCGACAGAC ACGTTGGATGACTCGAGACAACCTACAAGG 2086832 ACGTTGGATGCTTTTGAGCATCACATTCCTC ACGTTGGATGTGCCTAAGCACTGTATAACC 2129108 ACGTTGGATGAACTCCCAGTAAGTCCTTCC ACGTTGGATGACTCAGGCAGTAACTCCAAC 2129111 ACGTTGGATGTACACTTTTCCCGCAAGACC ACGTTGGATGGTCATGGACATCTACAGTATC 2170850 ACGTTGGATGGAAGGCCAATGCAAGGATAC ACGTTGGATGAAGAACACACAAAAAAAT 2279472 ACGTTGGATGGAGAAGAGCATTGGTTGCTG ACGTTGGATGTGCCCACAAGTGCTATCTAC 2291304 ACGTTGGATGGTCTCAGGAAGGTTTAGAGG ACGTTGGATGAAAAGACAAACGATATGGCC 2291305 ACGTTGGATGCATGATTTCAAAATCATGTTC ACGTTTGGATGGAGATGTACAGTATGAGTCC 2291306 ACGTTGGATGCAGCGACTAGTCATTTAACCG ACGTTGGATGCAGTTGGTTTCAACTCTGCC 2291309 ACGTTTGGATGCATTGTTGTTCCTACCATTC ACGTTGGATGAAAGTGGTAAAGGAGAGGCG 2291310 ACGTTGGATGGTGCTTGATACTTGGCCTAC ACGTTGGATGCAACTGGAAATTGCCGAAGC 2291311 ACGTTGGATGTCAACATTTTACTCCTAGCTC ACGTTGGATGATTTTGGGCTGTGGTCTTCC 2291312 ACGTTGGATGTGTATTCTCCTGCATCGCTC ACGTTGGATGTCCAAGTTCAAGAACGACAC 2291313 ACGTTGGATGTTCGAGTTTTACCGTATGGTG ACGTTGGATGGATCACAGACAGGTCAGTTG 2306636 ACGTTGGATGCTGAGACCAGTCTGTGTTTG ACGTTGGATGGTTTCCCATGACACTGTTCC 2306637 ACGTTGGATGCTACTACTATTTCTGGAGTC ACGTTGGATGCTTATGCATTTCAACTGCCAC 2366911 ACGTTGGATGGTAGATGCTTGAATCAATAAAG ACGTTGGATGATAGCAGCTCCAGAACTAGG 2366912 ACGTTTGGATGGAACTGTTGTTGAATGGGAC ACGTTGGATGCAATACTTGTAAAATAGCAGC 2366913 ACGTTGGATGCTATCTGTATTCTCATGGCTG ACGTTGGATGTTACCTAGTTCTGGAGCTGC 3769858 ACGTTGGATGCTACATGTCCATGGTTTGATG ACGTTGGATGGCATCAACCTTTATGCCAAG 3769860 ACGTTGGATGGTATACAGAATATTGCATGCC ACGTTGGATGGAACATCATTGAAGGTAAAG 3769863 ACGTTGGATGCAAGGATTTATTTACATGCTG ACGTTGGATGGTCATCAGGAGAAAGTAAGC 3816782 ACGTTGGATGGAGGAAACCAGAGCTTCAAG ACGTTGGATGCAGCACGCTGTTTCTCAATG 3816849 ACGTTGGATGAACCAGCTCACCTCAGGAAC ACGTTGGATGTTTGTGGTGCCCATTCAAAC

TABLE 31 dbSNP Extend Term rs# Primer Mix 726215 TTGAGAACACAGGATGC ACT 966783 CTCCCATTTTGGTCTTG ACG 998329 GGTGTTTTGTCAGTACAATT ACG 1387472 ACTACAAACTCTTTCCTTACC CGT 1484119 GTTGTTTATGTTTATGTTATGTGTT ACT 1484120 TGTGCCTCAGTTTCTCC CGT 1489479 GACAGCTGTAATTGTAGACC ACT 1489480 CTCAATCACATTTACCCTC ACT 1489481 TCTGATTGTTCCATTTAATATCTG ACT 1489482 CAGCTATCTGGAAATCTTGTTTGA CGT 1489483 GTGTACACATATAGCAACCTCA ACT 1489486 CTCTGACCTGTGAGCTAC ACT 1552280 GCTGTTGAATCAGGATTTGATT ACG 1565288 GGCAAAGAAACACTAGAAA ACG 1844334 CAGTTCGGCAGTTTCTT ACT 1872203 AAAAAATCATGAAAAGGAGCATG CGT 1905520 ACAAGTCTTTTCATGGTC ACG 2029395 CAAAATGAAGGAACACTTATCA ACG 2029397 AGCTCTGTTGGCACTTT ACT 2046777 GAGCCTGATTATTTGTTTGGGTA ACG 2046778 CTGTCATGATTGACAGGTCC ACT 2054708 CCTGGGCCTGGAAGGCAAC ACG 2078403 GGCTGGAGCAAGAATTA ACG 2086832 CAATGTAATCCTTGGATAGAT CGT 2129108 CAACTACATAGTCAGACTTT ACT 2129111 TATACGCAATAGTTCCTGGG ACT 2170850 GAACACACAAAAAAATTTAATCA ACT 2279472 CTCTTTAAACCTGCATTTTC ACT 2291304 CGATATGGCCATTTTGG ACT 2291305 CATATTCACACAATGGGAAAA CGT 2291306 CTGCCAACTATCAGCTT ACT 2291309 GCGAGACCATGGCATATAACA ACT 2291310 AACTTACACGTTTGTTGCTA ACT 2291311 GTGGTCTTCCGGATATCA ACG 2291312 CGACACAAATATGTAGTGGA ACT 2291313 TGTCTTGCTACATTCCAGT ACT 2306636 TCCAGTAAAATGGTTCCATAAGA ACT 2306637 TCAACTGCCACAAAATG ACT 2366911 TTCCTTTGTCCCATTCA ACT 2366912 TGTAAAATAGCAGCTCCAGAA CGT 2366913 ATTCTAAATGGAAAAAGAGCCA ACG 3769858 TGCCCTGAATGTGCCTC ACT 3769860 GGATAAGCATATGTAACTTTACG CGT 3769863 AAGTAAAAAGGACATAAAAACCT ACT 3816782 GTTGATGGAACAACATAAAA CGT 3816849 GCCCATTCAAACATAAAG ACT Genetic Analysis and Allelotyping Results

Allelotyping results are shown for cases and controls in Table 32. The allele frequency for the A2 allele is noted in the fifth and sixth columns for breast cancer pools and control pools, respectively, where “AF” is allele frequency. The allele frequency for the A1 allele can be easily calculated by subtracting the A2 allele frequency from 1 (A1 AF=1−A2 AF). For example, the SNP rs2291309 has the following case and control allele frequencies: case A1 (T)=0.190; case A2 (G)=0.810; control A1 (T)=0.215; and control A2 (G)=0.785, where the nucleotide is provided in paranthesis. SNPs with blank allele frequencies were untyped. TABLE 32 dbSNP Position in Chromosome A1/A2 A2 Control rs# Position Allele A2 Case AF AF p-Value 2291309 200 179587600 T/G 0.785 0.810 0.3114 2291310 381 179587781 T/C 0.069 0.073 0.8256 1484119 5303 179592703 G/C 0.726 0.714 0.6532 TTN_SNP1 6084 179593484 C/T 0.795 0.796 0.9735 1484120 6879 179594279 T/A 0.952 0.957 0.6579 2291312 7837 179595237 T/C 0.064 0.062 0.8817 3816782 7985 179595385 C/A 0.853 0.858 0.8139 2291313 9333 179596733 T/C 0.702 0.662 0.1474 2306636 11559 179598959 T/C 0.921 0.929 0.6434 2291304 12473 179599873 T/C 0.050 0.025 0.0307 2291305 12880 179600280 T/A 0.040 0.037 0.7733 1905520 13606 179601006 C/T 0.014 0.027 0.1559 2291306 14861 179602261 A/G 0.805 0.839 0.1348 TTN_SNP2 20658 179608058 C/T 0.773 0.821 0.0491 2054708 22200 179609600 G/A 0.893 0.883 0.6092 2306637 24525 179611925 A/C 0.999 0.956 0.0000 3769863 26373 179613773 T/G 0.382 0.409 0.3545 3769860 42869 179630269 A/T 0.025 0.022 0.7358 3816849 43713 179631113 A/G 0.374 0.390 0.5938 3769858 44429 179631829 A/G 0.515 0.561 0.1296 2279472 49037 179636437 A/G 0.916 0.871 0.0156 2046778 49170 179636570 A/G 0.171 0.231 0.0122 1565288 50206 179637606 G/A 0.978 0.979 0.9096 2129108 51552 179638952 C/T 0.886 0.807 0.0005 2170850 51674 179639074 T/G 0.906 0.863 0.0258 2029397 56427 179643827 T/C 0.094 0.114 0.2585 2029395 56844 179644244 G/A 0.825 0.810 0.5200 1844334 57953 179645353 A/G 0.536 0.533 0.9266  998329 60862 179648262 G/A 0.035 0.055 0.1078 1489486 61606 179649006 T/C 0.646 0.674 0.3300 2046777 62560 179649960 G/A 0.910 0.875 0.0573 1489483 65078 179652478 A/G 0.188 0.205 0.4857 1489482 65155 179652555 G/T 0.058 0.084 0.0944 2366911 70295 179657695 T/C 0.298 0.268 0.2753 2366912 70335 179657735 G/T 0.238 0.186 0.0341 2366913 70398 179657798 C/T 0.259 0.214 0.0772 2078403 79233 179666633 C/T 0.551 0.564 0.6795 1489481 80025 179667425 C/G 0.270 0.251 0.4774 2129111 84521 179671921 A/G 0.324 0.256 0.0127  966783 84540 179671940 C/T 0.210 0.178 0.1714 1489480 85170 179672570 T/G 0.426 0.398 0.3465 1489479 85300 179672700 A/C 0.414 0.372 0.1553  726215 87596 179674996 A/C 0.141 0.194 0.0173 1387472 89696 179677096 C/A 0.106 0.097 0.6242 2086832 92219 179679619 A/T 0.211 0.298 0.0014 1872203 96589 179683989 A/T 0.652 0.577 0.0113

FIG. 18 shows the proximal SNPs in and around the TTN region for females. The position of each SNP on the chromosome is presented on the x-axis. The y-axis gives the negative logarithm (base 10) of the p-value comparing the estimated allele in the case group to that of the control group. The minor allele frequency of the control group for each SNP designated by an X or other symbol on the graphs in FIG. 18 can be determined by consulting Table 32. By proceeding down the Table from top to bottom and across the graphs from left to right the allele frequency associated with each symbol shown can be determined.

To aid the interpretation, multiple lines have been added to the graph. The broken horizontal lines are drawn at two common significance levels, 0.05 and 0.01. The vertical broken lines are drawn every 20 kb to assist in the interpretation of distances between SNPs. Two other lines are drawn to expose linear trends in the association of SNPs to the disease. The light gray line (or generally bottom-most curve) is a nonlinear smoother through the data points on the graph using a local polynomial regression method (W. S. Cleveland, E. Grosse and W. M. Shyu (1992) Local regression models. Chapter 8 of Statistical Models in S eds J. M. Chambers and T. J. Hastie, Wadsworth & Brooks/Cole.). The black line (or generally top-most curve, e.g., see peak in left-most graph just to the left of position 92150000) provides a local test for excess statistical significance to identify regions of association. This was created by use of a 10 kb sliding window with I kb step sizes. Within each window, a chi-square goodness of fit test was applied to compare the proportion of SNPs that were significant at a test wise level of 0.01, to the proportion that would be expected by chance alone (0.05 for the methods used here). Resulting p-values that were less than 10⁻⁸ were truncated at that value.

Finally, the gene or genes present in the loci region of the proximal SNPs as annotated by Locus Link (http address: www.ncbi.nlm.nih.gov/LocusLink/) are provided on the graph. The exons and introns of the genes in the covered region are plotted below each graph at the appropriate chromosomal positions. The gene boundary is indicated by the broken horizontal line. The exon positions are shown as thick, unbroken bars. An arrow is place at the 3′ end of each gene to show the direction of transcription.

Example 8 In Vitro Production of Target Polypeptides

cDNA is cloned into a pIVEX 2.3-MCS vector (Roche Biochem) using a directional cloning method. A cDNA insert is prepared using PCR with forward and reverse primers having 5′ restriction site tags (in frame) and 5-6 additional nucleotides in addition to 3′ gene-specific portions, the latter of which is typically about twenty to about twenty-five base pairs in length. A Sal I restriction site is introduced by the forward primer and a Sma I restriction site is introduced by the reverse primer. The ends of PCR products are cut with the corresponding restriction enzymes (i.e., Sal I and Sma I) and the products are gel-purified. The pIVEX 2.3-MCS vector is linearized using the same restriction enzymes, and the fragment with the correct sized fragment is isolated by gel-purification. Purified PCR product is ligated into the linearized pIVEX 2.3-MCS vector and E. coli cells transformed for plasmid amplification. The newly constructed expression vector is verified by restriction mapping and used for protein production.

E. coli lysate is reconstituted with 0.25 ml of Reconstitution Buffer, the Reaction Mix is reconstituted with 0.8 ml of Reconstitution Buffer; the Feeding Mix is reconstituted with 10.5 ml of Reconstitution Buffer; and the Energy Mix is reconstituted with 0.6 ml of Reconstitution Buffer. 0.5 ml of the Energy Mix was added to the Feeding Mix to obtain the Feeding Solution. 0.75 ml of Reaction Mix, 50 μl of Energy Mix, and 10 μg of the template DNA is added to the E. coli lysate.

Using the reaction device (Roche Biochem), 1 ml of the Reaction Solution is loaded into the reaction compartment. The reaction device is turned upside-down and 10 ml of the Feeding Solution is loaded into the feeding compartment. All lids are closed and the reaction device is loaded into the RTS500 instrument. The instrument is run at 30° C. for 24 hours with a stir bar speed of 150 rpm. The pIVEX 2.3 MCS vector includes a nucleotide sequence that encodes six consecutive histidine amino acids on the C-terminal end of the target polypeptide for the purpose of protein purification. Target polypeptide is purified by contacting the contents of reaction device with resin modified with Ni²⁺ ions. Target polypeptide is eluted from the resin with a solution containing free Ni²⁺ ions.

Example 9 Cellular Production of Target Polypeptides

Nucleic acids are cloned into DNA plasmids having phage recombination cites and target polypeptides are expressed therefrom in a variety of host cells. Alpha phage genomic DNA contains short sequences known as attP sites, and E. coli genomic DNA contains unique, short sequences known as attB sites. These regions share homology, allowing for integration of phage DNA into E. coli via directional, site-specific recombination using the phage protein Int and the E. coli protein IHF. Integration produces two new att sites, L and R, which flank the inserted prophage DNA. Phage excision from E. coli genomic DNA can also be accomplished using these two proteins with the addition of a second phage protein, Xis. DNA vectors have been produced where the integration/excision process is modified to allow for the directional integration or excision of a target DNA fragment into a backbone vector in a rapid in vitro reaction (Gateway™ Technology (Invitrogen, Inc.)).

A first step is to transfer the nucleic acid insert into a shuttle vector that contains attL sites surrounding the negative selection gene, ccdB (e.g. pENTER vector, Invitrogen, Inc.). This transfer process is accomplished by digesting the nucleic acid from a DNA vector used for sequencing, and to ligate it into the multicloning site of the shuttle vector, which will place it between the two attL sites while removing the negative selection gene ccdB. A second method is to amplify the nucleic acid by the polymerase chain reaction (PCR) with primers containing attB sites. The amplified fragment then is integrated into the shuttle vector using Int and IHF. A third method is to utilize a topoisomerase-mediated process, in which the nucleic acid is amplified via PCR using gene-specific primers with the 5′ upstream primer containing an additional CACC sequence (e.g., TOPO® expression kit (Invitrogen, Inc.)). In conjunction with Topoisomerase I, the PCR amplified fragment can be cloned into the shuttle vector via the attL sites in the correct orientation.

Once the nucleic acid is transferred into the shuttle vector, it can be cloned into an expression vector having attR sites. Several vectors containing attR sites for expression of target polypeptide as a native polypeptide, N-fusion polypeptide, and C-fusion polypeptides are commercially available (e.g., pDEST (Invitrogen, Inc.)), and any vector can be converted into an expression vector for receiving a nucleic acid from the shuttle vector by introducing an insert having an attR site flanked by an antibiotic resistant gene for selection using the standard methods described above. Transfer of the nucleic acid from the shuttle vector is accomplished by directional recombination using Int, IHF, and Xis (LR clonase). Then the desired sequence can be transferred to an expression vector by carrying out a one hour incubation at room temperature with Int, IHF, and Xis, a ten minute incubation at 37° C. with proteinase K, transforming bacteria and allowing expression for one hour, and then plating on selective media. Generally, 90% cloning efficiency is achieved by this method. Examples of expression vectors are pDEST 14 bacterial expression vector with att7 promoter, pDEST 15 bacterial expression vector with a T7 promoter and a N-terminal GST tag, pDEST 17 bacterial vector with a T7 promoter and a N-terminal polyhistidine affinity tag, and pDEST 12.2 mammalian expression vector with a CMV promoter and neo resistance gene. These expression vectors or others like them are transformed or transfected into cells for expression of the target polypeptide or polypeptide variants. These expression vectors are often transfected, for example, into murine-transformed a adipocyte cell line 3T3-L1, (ATCC), human embryonic kidney cell line 293, and rat cardiomyocyte cell line H9C2.

Example 10 Haplotype Analysis of the GP6 Locus

rs1671152 is significantly associated with breast cancer at the allele level (P<0.05). This relationship does not hold at the genotype level. Very weak LD is observed across markers in the region. Estimated chi-squared statistics, odds ratios, and score tests indicate that haplotypes are not significantly associated with breast cancer.

Statistics

Chi-squared statistics are estimated to assess whether 1) alleles and genotypes are associated with breast cancer status and 2) marker genotype frequencies deviate significantly from Hardy-Weinberg equilibrium (HWE). Haplotype frequencies and relative frequencies are estimated, as well as several statistics (r², D′, and p-value) that gauge the extent and stability of linkage disequilibrium between markers in each region. Chi-squared statistics and score tests are estimated to determine whether reconstructed haplotypes are significantly associated with breast cancer status (P <0.05). P-values are estimated for 1) the full set of reconstructed haplotypes and 2) a reduced set that excludes haplotypes with observed frequencies less than 10. Results are presented by chromosome order.

Results

Summary Statistics: Alleles and Genotypes SNP Locations SNP.ID Type Location 269911 Proximal 60156885 1671152 Incident 60202366 2124090 Proximal 60240477

Allele by GYNGroup Case Control Test N (N = 508) (N = 536) Statistic 269911: T 1008 18% (90) 16% (85) Chi-square = 0.77 d.f. = 1 P = 0.38 1671152: G 1008 86% (408) 81% (431) Chi-square = 3.98 d.f. = 1 P = 0.0462 2124090: A 1022 13% (64) 11% (56) Chi-square = 1.47 d.f. = 1 P = 0.226

Genotype by GYNGroup Case Control N (N = 254) (N = 268) Test Statistic 269911: AA 504 68% (165) 71% (184) Chi-square = 0.73 d.f. = 2 P = 0.693 AT 28% (68) 26% (67) TT  5% (11)  3% (9) 1671152: TT 504  4% (10)  5% (13) Chi-square = 4.79 d.f. = 2 P = 0.0912 TG 20% (48) 28% (75) GG 76% (180) 67% (178) 2124090: CC 511 75% (184) 80% (213) Chi-square = 3.42 d.f. = 2 P = 0.181 CA 24% (60) 18% (48) AA  1% (2)  2% (4)

Genotyping QC: Test of Hardy-Weinberg Proportions All A.freq D ChiSq Pvalue 269911 0.824 0.00943 2.070 0.15000 1671152 0.832 0.01830 8.430 0.00369 2124090 0.882 −0.00167 0.128 0.72100

Control A.freq D ChiSq Pvalue 269911 0.836 0.00782 0.842 0.359 1671152 0.807 0.01290 1.780 0.182 2124090 0.896 0.00458 0.622 0.430

Summary Statistics: Linkage Disequilibrium PHASE Haplotype Frequencies H.freq H.relfreq AGA 113 0.115 AGC 534 0.542 ATA 1 0.001 ATC 164 0.166 TGA 2 0.002 TGC 171 0.173 TTC 1 0.001

Linkage Disequilibrium Between Markers r² 269911 1671152 2124090 269911 1.0000 0.0405 0.0233 1671152 0.0405 1.0000 0.0243 2124090 0.0233 0.0243 1.0000

D′ 269911 1671152 2124090 269911 1.000 0.207 0.193 1671152 0.207 1.000 0.192 2124090 0.193 0.192 1.000

P-value 269911 1671152 2124090 269911 1.00e+00 2.67e−10 1.68e−06 1671152 2.67e−10 1.00e+00 9.84e−07 2124090 1.68e−06 9.84e−07 1.00e+00

Haplotype by GYNGroup PHASE Haplotypes (All) Case Case (%) se.X{circumflex over ( )}2 Control Control (%) Control.X{circumflex over ( )}2 OR ln.OR ATC 66 6.72 1.80 98 9.98 1.62 0.6499 −0.4309 AGC 253 25.76 0.00 281 28.62 0.00 0.8658 −0.1441 TGC 87 8.86 0.42 84 8.55 0.38 1.0392 0.0385 AGA 60 6.11 0.76 53 5.40 0.68 1.1407 0.1316 Pearson Chi-squared Test = 5.6672, DF = 3, P-value = 0.129 Permutation Test P-value = 0.07

haplo.score Haplotypes Hap.Freq Score P.X{circumflex over ( )}2 P.Sim ATC 0.1647 −1.8903 0.0587 0.0599 AGC 0.5464 −0.0089 0.9929 0.9960 TGC 0.1695 0.8924 0.3722 0.3749 AGA 0.1104 1.2311 0.2183 0.2106 TGA 0.0053 2.2174 0.0266 0.0105 Global Score = 10.7498, DF = 5, Global P.X{circumflex over ( )}2 = 0.0566, Global P.Sim = 0.0425

Example 11 Haplotype Analysis of the CHGB Locus

rs454422 and rs236108 are significantly associated at the allele and genotype levels (P<0.05). Moderate LD is observed between 454422 and 236108 (r²=0.474). Chi-squared statistics indicate that haplotypes are significantly associated with breast cancer. Haplotype-specific chi-squared values, odds ratios, and score tests indicate that the TAT haplotype contributes most to this effect, suggesting that individuals who carry this haplotype are at a slightly lower risk of developing breast cancer than individuals with other haplotypes.

Statistics

Chi-squared statistics are estimated to assess whether 1) alleles and genotypes are associated with breast cancer status and 2) marker genotype frequencies deviate significantly from Hardy-Weinberg equilibrium (HWE). Haplotype frequencies and relative frequencies are estimated, as well as several statistics (r², D′, and p-value) that gauge the extent and stability of linkage disequilibrium between markers in each region. Chi-squared statistics and score tests are estimated to determine whether reconstructed haplotypes are significantly associated with breast cancer status (P<0.05). P-values are estimated for 1) the full set of reconstructed haplotypes and 2) a reduced set that excludes haplotypes with observed frequencies less than 10. Results are presented by chromosome order.

Results

Summary Statistics: Alleles and Genotypes SNP Locations SNP.ID Type Location 236116 Proximal 5884335 454422 Incident 5891693 236108 Proximal 5916559

Allele by GYNGroup Case Control Test N (N = 508) (N = 536) Statistic 236116: T 1028 79% (392) 79% (420) Chi-square = 0.08 d.f. = 1 P = 0.783 454422: C 998 83% (402) 76% (393) Chi-square = 8.06 d.f. = 1 P = 0.00452 236108: T 1016 8% (41) 13% (70)  Chi-square = 6.14 d.f. = 1 P = 0.0132

Genotype by GYNGroup Case Control Test N (N = 254) (N = 268) Statistics 236116: CC 514 4% (9) 3% (9) Chi-square = 0.22 d.f. = 2 P = 0.894 CT 34% (84) 36% (96) TT  62% (154)  61% (162) 454422: AA 499 3% (8)  5% (14) Chi-square = 8.37 d.f. = 2 P = 0.0152 AC 27% (64) 37% (95) CC  70% (169)  58% (149) 236108: CC 508  84% (206)  75% (198) Chi-square = 7.04 d.f. = 2 P = 0.0296 CT 14% (35) 23% (62) TT 1% (3) 2% (4)

Genotyping QC: Test of Hardy-Weinberg Proportions All A.freq D ChiSq Pvalue 236116 0.790 −0.00948 1.620 0.203 454422 0.798 0.00356 0.240 0.624 236108 0.893 0.00266 0.381 0.537

Control A.freq D ChiSq Pvalue 236116 0.789 −0.01320 1.6200 0.203 454422 0.762 −0.00209 0.0340 0.854 236108 0.869 −0.00150 0.0445 0.833

Summary Statistics: Linkage Disequilibrium PHASE Haplotype Frequencies H.freq H.relfreq CCC 207 0.210 TAC 94 0.095 TAT 106 0.107 TCC 581 0.588

Linkage Disequilibrium Between Markers r² 236116 454422 236108 236116 1.0000 0.0673 0.0319 454422 0.0673 1.0000 0.4740 236108 0.0319 0.4740 1.0000

D′ 236116 454422 236108 236116 1.000 0.265 0.265 454422 0.265 1.000 1.000 236108 0.265 1.000 1.000

D′ P-value 236116 454422 236108 236116 1.00e+00 3.33e−16 2.02e−08 454422 3.33e−16 1.00e+00 0.00e+00 236108 2.02e−08 0.00e+00 1.00e+00

Haplotype by GYNGroup PHASE Haplotypes (All) Case Case(%) Case.X{circumflex over ( )}2 Control Control(%) Control.X{circumflex over ( )}2 OR ln.OR TAT 39 3.95 2.85 67 6.78 2.65 0.5649 −0.5711 TAC 39 3.95 0.87 55 5.57 0.81 0.6971 −0.3608 CCC 99 10.02 0.01 108 10.93 0.00 0.9074 −0.0972 TCC 299 30.26 1.30 282 28.54 1.21 1.0864 0.0829 Pearson Chi-squared Test = 9.7095, DF = 3, P-value = 0.02120

PHASE Haplotypes (Low Frequency Excluded) Case Case(%) Case.X{circumflex over ( )}2 Control Control(%) Control.X{circumflex over ( )}2 OR ln.OR TAT 39 4.36 3.17 67 7.49 3.03 0.5630 −0.5745 CCC 99 11.07 0.05 108 12.08 0.05 0.9063 −0.0984 TCC 299 33.45 0.79 282 31.54 0.76 1.0906 0.0867 Pearson Chi-squared Test = 7.8414, DF = 2, P-value = 0.01983

haplo.score Haplotypes Hap.Freq Score P. X{circumflex over ( )}2 P.Sim TAT 0.1073 −2.4492 0.0143 0.0130 TAC 0.0951 −1.3720 0.1701 0.1880 CCC 0.2095 −0.1174 0.9065 0.9372 TCC 0.5881 2.4468 0.0144 0.0145

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the invention, as set forth in the claims which follow. All publications or patent documents cited in this specification are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.

Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. U.S. patents, documents and other publications referenced herein are hereby incorporated by reference. 

1. A method for identifying a subject at risk of breast cancer, which comprises detecting the presence or absence of one or more polymorphic variations associated with breast cancer in a nucleic acid sample from a subject, wherein the one or more polymorphic variations are detected in a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence in SEQ ID NO: 2; (b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 2; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NO, 2; (d) a fragment of a nucleotide sequence of (a), (b), or (c); whereby the presence of the polymorphic variation is indicative of the subject being at risk of breast cancer.
 2. The method of claim 1, which further comprises obtaining the nucleic acid sample from the subject. 3-5. (canceled)
 6. The method of claim 1, wherein the one or more polymorphic variations are detected at one or more positions in SEQ ID NO: 2 selected from the group consisting of 184, 506, 3981, 7815, 7875, 10775, 10786, 11013, 11020, 11101, 14171, 14278, 16512, 16706, 18442, 20286, 21591, 22275, 25318, 27997, 29840, 31088, 31258, 32367, 32427, 33671, 38796, 41530, 41874, 44161, 47502, 51089, 51205, 53645, 54280, 57610, 57740, 60812, 60837, 64448, 65249, 65482, 66535, 66789, 67214, 68347, 69060, 70100, 70215, 73687, 73732, 74183,74813, 78136, 79540, 79655, 79731, 82111, 82155, 83479, 84511, 85290, 90620, 91127, 92095, 92679, 94839 and
 95220. 7. The method of claim 1, wherein the one or more polymorphic variations are detected at one or more positions in SEQ ID NO: 2 selected from the group consisting of 506, 3981, 7815, 7875, 11020, 11101, 18442, 47502, 53645, 65249, 73687, 73732, 74183, 79540, 82155, 85290, 90620, 91127, 92095, 92679, 94839 and
 95220. 8. The method of claim 1, wherein the one or more polymorphic variations are detected at one or more positions in a region spanning positions 506-95220 in SEQ ID NO:
 2. 9-17. (canceled)
 18. The method of claim 1, wherein the one or more polymorphic variations are detected at one or more positions in linkage disequilibrium with one or more positions in claim
 6. 19. The method of claim 1, wherein detecting the presence or absence of the one or more polymorphic variations comprises: hybridizing an oligonucleotide to the nucleic acid sample, wherein the oligonucleotide is complementary to a nucleotide sequence in the nucleic acid and hybridizes to a region adjacent to the polymorphic variation; extending the oligonucleotide in the presence of one or more nucleotides, yielding extension products; and detecting the presence or absence of a polymorphic variation in the extension products.
 20. The method of claim 1, wherein the subject is a human, 21-50. (canceled)
 51. A method for detecting or preventing breast cancer in a subject, which comprises: detecting the presence or absence of one or more polymorphic variations associated with breast cancer in a nucleic acid sample from a subject, wherein the polymorphic variation is detected in a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence in SEQ ID NO: 2; (b) a nucleotide sequence which encodes a polypeptide encoded by a nucleotide sequence in SEQ ID NO: 2; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to the amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 2; (d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic variation; and administering a breast cancer prevention procedure or detection procedure to a subject in need thereof based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.
 52. The method of claim 48, wherein the one or more polymorphic variations are detected at one or more positions in wherein the one or more polymorphic variations are detected at one or more positions in claim
 6. 53. The method of claim 48, wherein the breast cancer detection procedure is selected from the group consisting of a mammography, an early mammography program, a frequent mammography program, a biopsy procedure, a breast biopsy and biopsy from another tissue, a breast ultrasound and optionally ultrasound analysis of another tissue, breast magnetic resonance imaging (MRI) and optionally MRI analysis of another tissue, electrical impedance (T-scan) analysis of breast and optionally of another tissue, ductal lavage, nuclear medicine analysis (e.g., scintimammography), BRCA1 and/or BRCA2 sequence analysis results, thermal imaging of the breast and optionally of another tissue, and a combination of the foregoing.
 54. The method of claim 48, wherein the breast cancer prevention procedure is selected from the group consisting of one or more selective hormone receptor modulators, one or more compositions that prevent production of hormones, one or more hormonal treatments, one or more biologic response modifiers, surgery, and drugs that delay or halt metastasis.
 55. The method of claim 51, wherein the selective hormone receptor modulator is selected from the group consisting of tamoxifen, reloxifene, and toremifene, the composition that prevents production of hormones is an aramotase inhibitor selected from the group consisting of exemestane, letrozole, anastrozol, groserelin, and megestrol; the hormonal treatment is selected from the group consisting of goserelin acetate and filvestrant; the biologic response modifier is an antibody that specifically binds herceptin/HER2; the surgery is selected from the group consisting of lumpectomy and mastectomy; and the drug that delays or halts metastasis is pamidronate disodium. 56-58. (canceled)
 59. A method of selecting a subject that will respond to a treatment of breast cancer, which comprises: detecting the presence or absence of one or more polymorphic variations associated with breast cancer in a nucleic acid sample from a subject, wherein the polymorphic variation is detected in a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 2; (b) a nucleotide sequence which encodes a polypeptide consisting of an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 2; (c) a nucleotide sequence which encodes a polypeptide that is 90% or more identical to an amino acid sequence encoded by a nucleotide sequence in SEQ ID NO: 2; and (d) a fragment of a nucleotide sequence of (a), (b), or (c) comprising the polymorphic variation; and selecting a subject that will respond to the breast cancer treatment based upon the presence or absence of the one or more polymorphic variations in the nucleic acid sample.
 60. The method of claim 56, wherein the one or more polymorphic variations are at one or more positions in wherein the one or more polymorphic variations are detected at one or more positions in claim
 6. 61-64. (canceled) 